| Objective1 To develop an intergrated clinical virology molecular diagnostic platform which combined a series of PCR technology including PCR, real-time PCR and multiplex PCR with high throughput sequencing technology, in order to screening viral pathogens rapidly and dealing with emergence public health problem by its capacity of discovering novel viral pathogen in hospital and disease control centers.2 To develop a respiratory viruses screening multiplex PCR system, a metagenomic high through-put sequencing platform and adenovirus detection and typing system, and to explore their application for clinical etiology research on pediatric respiratory system infection.3 Since large-scale dengue outbreak in Guangzhou,2014, a dengue virus (DENV) universal detection and typing PCR system has been developed, then we engage to understand viral molecular epidemiological characteristics and clinical features of dengue fever inpatients in our hospital. In addition, the values of molecularbiology method and immunology method used in laborartory diagnosis of dengue fever we also investigated.Methods1 Intergration and application of respiratory virus molecular diagnostic system1.1 Development of respiratory virus multiplex PCR screening system and its application in the viral spectrum research of pediatric acute rhinosinusitisA multiplex PCR panel for detection of Influenza A virus (IFA), Influenza B virus(IFB), Human parainfluenza virus (HPIV), Respiratory Syncytial virus (RSV), Adenovirus (ADV), Rhinovirus (RV), Enterovirus(EV), Human metapneumovirus (HMPV), Bocavirus, Coronavirus OC43, Coronaviruses NL63, Coronaviruses 229E was developed. A total of 259 acute rhinosinusitis (ARS) children,219 acute upper respiratory tract infection (AURI) children and 86 normal controls were recruited for the viral spectrum study. Chi-square test was used to analyze the relationship of these viruses with ARS.1.2 Development of respiratory metagenomic NGS platform used for the pathogen investigated of children severe pneumoniaA respiratory metagenomic next-generation sequencing (NGS) platform based on Ion Torrent/Proton sequencer was developed and used for etiology study in bronchoalveolar lavage fluid (BALF) samples from 9 patients. All viruses without prior knowledge would be identified. And the abundance for each kind od viral could be calculated. The whole genome of the dominant viral could be assembled by Trinity software bases on a large scale raw data. The SNPs or InDel mutations were analyzed in parallel through TVC and GATK method. Moreover, phylogenetic tree was constructed with BioEdit7.0 and MEGA6.0. The pathogens identified by NGS, PCR and traditional culture method was compared with each other.1.3 Development of adenovirus detection and typing system and the research on epidemic features of adenovirus subtypesUniversal and subtype specificity primer (probe) for adenovirus Hexon gene were designed. Total of 105 confirmed ADV positive nucleic samples were detected and subtyping by our PCR system. The distribution of genotypes of adenovirus in patients with respiratory infection was characterized.2 Development and evaluation of dengue fever molecular diagnostic method2.1 Development of dengue virus PCR and Real-time PCR detection system and the epidemiology analysis of dengue outbreak in Guangzhou,2014.Universal and typing primers (probes) for four dengue viruses were designed. Serum samples from laboratory confirmed DENV patients at the First Affiliated Hospital of Guangzhou Medical University were used for identification of virus subtype. In addition, genetic sequences of the envelope and non-structural genes from two isolates were analyzed for epidemiology study.2.2 Comparison of the performance of molecular and immunology diagnostic method for dengue diagnosisA total of 254 serum samples from 138 dengue fever patients confirmed with WHO criteria were detected by real-time PCR, NS1 antigen ELISA detection and anti-dengue virus IgM/IgG antibody test at the same time. The positive rate of each method was calculated by the day. Their tendency during dengue fever course was shown. A test recommendation for dengue fever diagnosis was summarized in this study.2.3 Analysis of clinical characteristics and laboratory test features for dengue patients in GuangzhouPatients were classified into dengue fever and severe dengue fever or DENV-1 or DENV-2 virus infected groups. The clinical characteristics and laboratory test features from these 138 dengue fever hospitalized patients were summarized and compared between diffirent groups.Results1 Intergration and application of respiratory virus molecular diagnostic system1.1 Development of respiratory virus multiplex PCR screening system and its application in the viral spectrum study of pediatric acute rhinosinusitisA total of 179 (31.7%) viral strains were identified with our in-house multiplex PCR panel. The viral detection rate in group ARS (44.4%), AURI (27.4%) and control (4.7%) were significantly different (RR=2.116,95%CI=1.440-3.110). Rhinovirus was the most prevalent virus,24 strains RV (9.3%) found in group ARS and 9 (4.1%) in group AURI (P<0.05, RR=2.27).16 samples (6.2%) were adenovirus positive in group ARS and 4 (1.6%) in group AURI (P<0.05, RR=3.374). The rest pathogens showed no significant difference between the two groups.1.2 Development of respiratory metagenomic NGS platform used for pathogen identification of children severe pneumoniaPathogens in BALF samples were successfully detected by metagenomic NGS sequencing technology. The results showed that the proportion of bacteria, fungi and viruses were different in individual sample. ADV-7 was the major pathogen in seven samples including one sample co-infected with HPIV. MPV and IFA (H3N2) virus was the major pathogen in another two samples respectively. Moreover, RSV, TTV, EBV, HPV and phages clould also be identified although they were in low abundance and coverage. Four ADV-7 full genome have obtained by de novo assemble, which were closely relative with nucleotide identities of> 99.7%. The pathogen identified by PCR was almost the same as by NGS, except that HPIV was lost in one case.In addition to a large number of normal floras, Haemophilus parainfluenzae, Escherichia coli, Mycoplasma Pneumoniae, Rothia mucilaginosa and Candida tropical is was detected simultaneously. Results obtained by traditional culture method were nearly the same as by NGS, while two or more bacteria co-infection would be found easily by the latter technology. As fungi detection, a Candida albicans was missed by NGS and Candida tropicalis was wrongly identified as Saccharomyces cerevisiae by traditional culture method.1.3 Development of adenovirus DNA detection and typing system and the study on epidemic features of adenovirus subtypeElectrophresis results were displayed single band in right size when samples amplified with ADV universal and typing primer respectively. All of 105 clincal samples with adenovirus positive could succeefully identified by ADV universal real-time PCR. However, only 82.9%(87) of them could be detected by PCR system. The most prevalent ADV serotypes were ADV-3 (57.5%). ADV-7 (26.4%), ADV-4 (5.7%) rate was followed in turns, ADV-14 and ADV-21 were both 1.1%. The infection of ADV-7 (66.7%) was higher than that of ADV-3 (33.3%) in severe pneumonia patients.2 Development and evaluation of dengue fever molecular diagnostic method2.1 Development of dengue virus PCR and real-time PCR detection system and the epidemiology analysis of dengue outbreak in Guangzhou,2014Total of 138 patients were recruited in our dengue study. Since viuse was failed to be detected in 14 patient,107 (77.5%) were serotype 1 and 17 (12.3%) were serotype 2.Phylogenetically, DENV-1 and DENV-2 were both highly similar to strains circulating in Guangdong province in recent years.2.2 Comparison of the performance of molecular and immunology diagnostic method for dengue fever diagnosisThe first time of fever onset was defined as day 1. Viral RNA detection rate was 100% before day 5 and over 80% until day 7. The positive rate of NS1 antigen detected by ELISA method maintained 90% by days 3-9. The positive rate of IgM antibodies increased to 80% by day 6, while that of IgG generally increase lowly before day 8 but still detectable in several months. Diagnosis rate could reach 100% with a combination of RNA and NS1 by days 1-8. The positive rate of antibody test was nearly 100% after day 10.2.3 Analysis of clinical characteristics and laboratory test features for dengue patients in GuangzhouPatients with dengue infection commonly showed typical clinical symptoms including fever (100%), aches/pains (52.2%), rash (31.2%) and digestive tract symptoms (20%-30%). Among the 8 severe cases,2 (25%) developed shock while 6 (75%) showed impaired consciousness. The majority of the patients showed leukopenia(2.85±1.6)/L and thrombocytopenia(78±51.8)/L, increased level of serum ALT, AST, LDH, D-Dimer and CK. There was no significant difference on clinical features or laboratory tests between the patients infected with serotype 1 or 2. The period of hospitalization of SDF patients was significantly longer than that of DF patients (p<0.05). The level of PLT(29.6 ± 27.2)/L, ALT (138.3±164.6)U/L, AST (179.5 ± 117.5)U/L, CK(1969.6 ± 3821.5) U/L, LDH (2743.3 ± 6148.4) U/L, D-Dimer (5113.7±3962.9) ng/ml, Cr (276.4 ± 426.6)umol/L and BUN(10.0±7.5)mmol/L in SDF patients was shown significantly different from DF patients.Conclusion1 Development and application of respiratory virus molecular diagnostic systemA fast, cheap and middle throughput respiratory virus screening multiplex PCR panel was developed in our study, which can not only meet the requirement of clinical diagnosis, but also is a very important pathogen screen tool for dealing with emergent public health problems. The novel or newly emergent viruses, undetectable by PCR, can be identified through metagenomic NGS platform in a short time. The methodology is independent of specific amplification. Moreover, the diversity and abundance of all microbial, full genome and mutations of the major virus can be characterized at the same time. The platform has posed promising on the diagnosis, academical research of clinical virology, which could be helpful in the pathogent tracing during acute infectious disease outbreak.We found that the risk of suffering ARS would be increased because of respiratory virus infection, especially RV and ADV. According to the etiology research of children severe pneumonia, ADV-7 has been found as a common viral pathogen and was always co-existed with bacteria or fungi. ADV-3 was the dominant adenovirus subtype in Guangzhou. The sequences of four ADV-7 strains were highly similar. ADV-7 had higher detection rate in severe pneumonia children, it is likely that ADV-7 was an important pathogen for children severe pneumonia, but the pathogenesis of ADV-7 is still unclear.2 Development and evaluation of dengue fever molecular diagnostic methodA dengue virus universal and typing PCR identify system has been developed. Two different dengue virus serotypes were identified co-circulating in Guangzhou epidemic,2014.Each of these serotypes is genetically highly related to viruses found in the Guangdong province in recent years. Given the high diversity and co-circulation of dengue viruses in endemic countries it is unlikely that the same strain of both serotypes was imported into Guangdong in each of these years. This observation, together with the size of the 2014 epidemic suggests the establishment of endemic transmission in Guangdong for both DENV-1 and DENV-2. Our results showed that there was no significant difference on clinical features or laboratory tests between the patients infected with serotype 1 or 2. While the platelet count of SDF patients was significantly lower than that of DF patients, and the function injury of multiple organs, the period of hospitalization of SDF patients was significantly different from DF patients too.Viral RNA detection was the best diagnosis in the early stage of DENV infection. NS1 antigen kept longer time in peripheral blood. While antibodies increased later. We suggest vRNA and NS1 test before day 8, NS1 and antibodies would be the better choice in the day 7-10. The combination of available test will be contributed to a right clinical diagnosis of dengue fever. |
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