| Objective: In order to develop a two-tube multiplex real-time quantitative RT-PCR assay for the detection of six respiratory paramyxovirus (human metapneumovirus, HMPV; respiratory syncytial virus subtype A, RSV-A; respiratory syncytial virus subtype B, RSV-B; parainfluenza virus type 1, PIV-1;parainfluenza virus type 2, PIV-2;parainfluenza virus type 3,PIV-3) and to evaluate a multiplex pathogen detection kit named RespirFinder?2SMART.Meanwhile, we analyzed the pathogen profiles and epidemiological characteristics of patients with acute respiratory infection(ARI) from Qinghai Province.Methods: Firstly, to establish a two-tube multiplex real time quantitative RT-PCR(qRT-PCR) assay, the primers and Taqman probes were designed and the reaction conditions were optimized Secondly, this novel qRT-PCR was used to detect 471 nasopharyngeal swabs (NPAs) from the sentinel surveillance hospital for febrile respiratory syndrome (FRS) in Qinghai Province followed by epidemiological analysis of six common human paramyxoviruses infections.Then, a commercial kit (RespirFinder?2SMART kit) were evaluated which allows simultaneous detection of' 22 respiratory pathogens, including 18 viruses and 4 bacteria. We did a parallel assay of the NPAs with this kit and 12 pre-established in-house assays (including multiple qRT-PCR for human coronavirus, qPCR for human adenovirus, qRT-PCR for human rhinovirus and multiple qRT-PCR for six human paramyxovirus) and then compared their results. The inconsistent results were further verified by nested PCR or quantitative real time PCR method or sequencing.Finally, the pathogen spectrum and epidemiological characteristics were analyzed for patients with ARI from Qinhai province.Results:Part one. Establishment and application of a novel multiplex real time quantitative RT-PCR for detecting six common human paramyxoviruses.The limit of detection of the six individual paramyxovirus in this novel multiplex qRT-PCR assay is 101copies/?l,corresponding to single-tube qRT-PCR assay, respectively. The linear range of the standard curve is 101copies/ul to 107copies/?l each paramyxovirus. There was no cross-reactivity with other respiratory virus including influenza A virus(InfA), influenza B virus (InfB),human coronaviruses (HCoV (-229E, -OC43, -NL63, -HKU1)), human rhinovirus (HRV),human adenovirus (HAdV) and human Bocavirus (HBoV). The coefficient of variation (CV) of the threshold value(Ct) was 3.95%. Detection results of 471 NPAs samples showed that the multiplex real time RT-PCR of six paramyxoviruses was in good agreement with its corresponding single-tube assay.Based on 445 NPAs samples with valid clinical information,the detection rates of HMPV, RSV-A, RSV-B, PIV-1, PIV-2 and PIV-3 were 0.44% (2/455), 3.15%(14/445) , 0.22% (1/445) , 2.70% (12/445),2.70% (12/445) and 2.02%(9/445),repectively. 42(9.44%) were positive for at least one paramyxovirus among 445 clinical samples, of which single infection was 35 and co-infection was 7.RSV-A (11,26.19%)was the most frequent detection in single infection samples,while either two dual-infection (3 cases, 7.14%) or triplex-infections (3 cases, 7.14%)were existed. Positive samples of each paramyxovirus were mainly distributed in less than 5 years age group and over 65 years' age group. The median age was 76 years old in patients with HMPV infection and 3 years old in patients with PIV-3 infection. There was no significant differences in the sex distribution. Moreover,PIV-3 and PIV-2 were mainly found in the spring. PIV-1, RSV-A and RSV-B were mainly found in winter. For most of the positive cases, the median body temperature is greater than 38 ?,but cases with RSV-B and HMPV infection was less than 38 ?.Part two. Evaluation and application of RespiFind?2SMART Multiple Pathogen Detection Kit.RespirFind 2SMART is a multiplex assay kit for respiratory pathogens covering 22 pathogens, including 18 viruses and 4 bacteria,with the characteristics of simple system configuration and short detection time. Based on the in-house assays established in our laboratory and 471 clinical samples from ARI, the RespirFind?2SMART Multiple Detection Kit and in-house assays were used for parallel detection and analysis of 12 respiratory viruses, including 4 human coronavirus (HCVV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1), human adenovirus (HAdV), human rhinovirus (HRV) and 6 common human paramyxoviruses (PIV-1, PIV- 3, HMPV, RSV-A and RSV-B). 22 pathogens contained in the RespirFind?2SMART Kit was all detected but the positive samples of each paramyxovirus were variable. Influenza A virus (InfA) is the most detected virus and Mycoplasma pneumoniae (M. pneu) is the most detected bacteria. The results of HMPV, HCoV-OC43, HCoV-HKU1, PIV-3 and RSV-B detected by RespirFind?2SMART kit was same as in-house assays, but different in RSV-A,PIV-1,PIV-2,HCoV-229E, HAdV and HRV. The positive samples of RSV-A(sensitivity,64.28%), PIV-1 (sensitivity,58.33%) and PIV-2(sensitivity,57.14%)detected by the kit were less 5 cases, 5 cases and 6 cases than that by in-house assays, respectively. The nested PCR method was used to confirm inconsistent samples of RSV-A, PIV-1 and PIV-2, with 5/5, 0/5 and 0/6 confirmed positive. The positive numbers of HCoV-229E (sensitivity available), HAdV (sensitivity, 100% )and HRV (sensitivity, 100%) tested by kit were higher than those of in house assay,with extra 2 cases, 13 cases and 17 cases,respectively. Inconsistent samples of HAdV and HRV were confirmed by nested PCR and sequencing, with 6/13 and 12/17 confirmed positive. The inconsistent samples of HCoV-229E were confirmed by real time RT-PCR, resulting of 1 positive case.The pathogen spectrum and epidemiology of 445 samples with valid clinical information were analyzed based on comprehensive results of in-house assays and the kit. The results showed that 226 (51%) samples were positive for pathogen infection. 147 (33%) samples were single infection. 55 (12%) samples were virus mixed infection and 24 (6%) samples were virus and bacterial mixed infection.Among 22 kinds of pathogens detected,IFV (InfA,InfB,HlN1pdm09)?HAdV?PIV?HCoV(-229E, -OC43, -HKU1,NL63 )?HRV/HEV and M.pneu were in front,with the detection rates of 22.64% (123/445), 12.58% (56/445), 8.76% (39/445),8.09%(36/445), 7.42% (33/445) and 6.29% (28/445) respectively. Legionella pneumophila (L.pneu) and B. pertussis (B.pert) had 1 case and 2 cases,respectively.The epidemiological characteristics of IFV, HCoV?, HAdV, HRV/EV and M.pneu were further analyzed. In addition to HAdV (13 years old), the median age of the other pathogens was greater than 18 years old. IFV was mainly distributed in the age group of 0?1 years old. HAdV was mainly distributed in the age group of 18?40 years old. HCoV is mainly distributed in the age group of 18?40 years. There was no significant difference in the detection rate between male and female or the outpatient and inpatient cases, except for HRV/HEV with statistically significant in outpatient and inpatient cases. Cough was mainly clinical symptoms for all patients with pathogen infections and the average body temperature were above 38?.Pathogens performed different distribution characteristics in different months.IFV peaked in January (11 cases), July (18 cases), and September (24 cases); HAdV had two relatively high detection peaks, which were located in January (8 cases) and December (10 cases). HRV/EV peaked in April (4 cases) and July (7 cases).M.pneu positive cases were mainly distributed in September, October and November. HCoV showed a significant peak,located in July, with a total of 11 cases. HRV/HEV had two peaks, located in April and July, with 4 cases and 7 cases, respectively, and the remaining months were relatively few.In conclusion. 1. In this study, a novel two-tube multiplex quantitative RT-PCR assay was established for detecting six common paramyxoviruses and the effect was similar to that of the corresponding single-tube assay applied for detection of 471 clinical samples, indicating that the novel assay has good sensitivity, specificity and stability. The detection of six paramyxoviruses in NPAs samples from Qinghai ARI population showed that the overall detection rate of six paramyxoviruses was 9.44%(42/445), of which RSV-A was the highest of 3.15% (14/445).2. The parallel comparison between RespirFind? 2SMART Kit and in-house assays for 12 respiratory viruses showed that the RespirFind?2SMART kit has a high sensitivity for detection of HCoV, HAdV and HRV, but with a lower sensitivity for detection of paramyxoviruses. All of 22 pathogens contained in the RespirFind?2SMART kit was detected from the NPAs samples from Qinghai Province.3. The pathogens detection from 455 NPAs samples of Qinghai with an positive results of 226 (51%). The most detected pathogen was influenza virus as 27.64%(123/445). Epidemiological analysis showed that different pathogens distributed in different age groups, and several individual viral infections occurred in different season. |
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