key: cord-0708070-7h2o7r4x authors: Liu, Gao‐Shan; Niu, Pei‐Hua; Zhao, Sheng‐Cang; Lu, Rou‐Jian; Tan, Wen‐Jie title: Detection of six common human paramyxoviruses in patients with acute febrile respiratory symptoms using a novel multiplex real‐time RT‐PCR assay date: 2018-11-19 journal: J Med Virol DOI: 10.1002/jmv.25350 sha: 6e267fde0c109902c70c5b1508d12b66523488c2 doc_id: 708070 cord_uid: 7h2o7r4x Human metapneumovirus (hMPV), respiratory syncytial virus type A (RSV‐A), RSV‐B, and human parainfluenza viruses 1, 2, and 3 (HPIV‐1, HPIV‐2, and HPIV‐3) are common respiratory paramyxoviruses. Here, we developed a two‐tube triplex one‐step real‐time reverse‐transcription polymerase chain reaction (real‐time RT‐PCR) and evaluated its performance using clinical samples. The data showed that this novel assay was 100% consistent with the monoplex real‐time RT‐PCR assay (in‐house), which was superior to the commercial routine multiplex‐ligation‐NAT‐based assay. Meanwhile, the clinical nasopharyngeal swabs of 471 patients with the acute febrile respiratory syndrome (AFRS) were analyzed using the established method. The results showed that 52 (11.7%) cases were positive for paramyxovirus. Among them, HPIVs and RSV‐A had the highest detection rate. The age and seasonal distribution of human paramyxovirus infection were analyzed. In conclusion, we developed a novel multiplex real‐time RT‐PCR assay for the rapid detection of six common human paramyxoviruses, which were dominant in patients with AFRS in Qinghai. Acute respiratory infections (ARIs) are common respiratory diseases that continue to pose a threat to public health. According to a report published by the World Health Organization, lower respiratory infections killed 3 million people and are the most deadly communicable disease, causing 3 million deaths worldwide in 2016 (http://www.who.int/mediacentre/factsheets/ fs310/en/index.html). More than 200 viruses are major etiological agents of ARIs. Respiratory syncytial virus (RSV), human metapneumoviruses (hMPVs), and human parainfluenza viruses (HPIVs) that are classified into the subfamilies Pneumovirinae and Paramyxovirinae and the Paramyxoviridae has a nonsegmented, single-stranded, negative-sense RNA genome, 1 which was the common causative agents of ARIs in humans in all age groups. [2] [3] [4] [5] These viruses can cause acute respiratory diseases such as croup, bronchiolitis, and pneumonia in children, the elderly, and immunodeficiency patients. RSV has two subtypes, subtype A and B, which play an important role in most respiratory infections and account for 60% to 80% of cases of bronchiolitis in children under 2 years of age. Followed by RSV, 5% to 15% of children with respiratory infections were hospitalized for bronchiolitis caused by hMPV. 6, 7 HPIVs have four subtypes including HPIV-1, HPIV-2, HPIV-3, and HPIV-4; HPIV-4 is considered less important. HPIV-1, HPIV-2, and HPIV-3 are prevalent in acute respiratory infections in children under 5 years and may account for 17% of hospitalizations. 5 RSV, hMPV, and HPIV infections can cause fever, cough, hypoxia, and severe symptoms such as bronchiolitis and pneumonia. 4, 8, 9 Therefore, it is difficult to distinguish between RSV, hMPV, and HPIV infections, and accurate early diagnosis based on clinical manifestations is a serious challenge. 10 Early diagnosis is recognized as an important way to facilitate early management and combat ARIs. 11 Real-time polymerase chain reaction (PCR) can detect and quantify specific DNA or RNA in samples. It is widely used to detect respiratory viruses and provides an effective solution for early detection. 12, 13 Compared with traditional virus culture and immunofluorescence detection methods, real-time PCR has the advantages of high sensitivity, short turnaround time, and simultaneous detection of multiple pathogens. Unlike monoplex real-time PCR, multiplex real-time PCR allows multiple pathogens to be detected simultaneously in a single reaction, thus having the advantage of time savings and costeffective. 7, 14 At present, several monoplex or multiplex real-time RT-PCR methods for hMPV, RSV, and HPIVs detection have been established. However, these techniques do not involve the simultaneous detection of all common respiratory paramyxoviruses (especially hMPV), and their performance and clinical evaluation are few. Here, we developed a one-step two-tube triplex real-time RT-PCR assay and assessed its sensitivity and specificity for hMPV, RSV-A, RSV-B, HPIV-1, HPIV-2, and HPIV-3. This new method was used to detect these viruses in 471 hospitalized patients with ARIs, and its performance was compared with a commercial routine multiplex- Here, we developed a two-tube triplex one-step real-time RT-PCR assay for simultaneous detection of all common respiratory paramyxoviruses. One tube is set for detection of HPIV-1, HPIV-2, and HPIV-3. The second tube is set for detection of RSV-A, RSV-B, and hMPV. The sequence information of primers and probes for each triplex real-time RT-PCR is presented in Supporting Information Table S1 , along with their target genes, reaction concentrations, and fluorescent-dye labels. We have screened and optimized six common respiratory paramyxovirus primers and probes from previous reports. 1, [15] [16] [17] [18] Primer Express software (version 3.0; Applied Biosystems, Foster City, CA) was used to modify sequences, and Oligo (version 7.57; Molecular Biology Insights, Colorado Springs, CO) was used to ensure that primer complements and primer dimers did not exist among different viruses in the same tube. Nuclease-free water was used as the negative control. The analytical sensitivity of the multiplex assay was determined by testing serial dilutions of the quantified RNA for each target. The specificity of the multiplex assay was determined by testing its cross- virus, the experiments were done in triplicate to obtain the coefficient of variation (CV). The linearity of triplex real-time RT-PCR was determined using GraphPad Prism 5 software (GraphPad). P < 0.05 was considered statistically significant. Figure S1 . Previous research has shown that primers and probes applied in triplex real-time RT-PCR perform with good specificity. 19 Meanwhile, cross-reactivity tests showed that the two-tube combinations were specific for each virus and there was no cross-reactivity with coronavirus, influenza A virus, influenza B virus, rhinoviruses, adenovirus, bocavirus, or echovirus. We used diethyl pyrocarbonate water as a negative control and the results showed that each tube did not cross-react among the three target viruses. Precision was estimated by performing the triplex real-time RT-PCR assay once per day during 3 days using the same protocol and same reagents. To assess intra-assay variation, three concentrations (10 7 , 10 5 , and 10 1 copies/µL) of each virus were tested five times per run. On the other hand, the same samples were tested five times in three separate runs to assess interassay variation. The results showed that the CVs were less than 3.04. The intraassay CVs ranged from 0.09 to 1.55, while the interassay CVs ranged from 0.69 to 3.04 (Supporting Information Table S2) A total of 471 clinical specimens from patients with AFRS were tested using triplex real-time RT-PCR and monoplex real-time RT-PCR. All target viruses were detected and total 55 were detected as positive (11.7%) for human paramyxoviruses from 42 clinical specimens among 471 clinical specimens. And HPIV-1, HPIV-2, and RSV-A were detected with the highest frequency, which 12 (2.54%), 14 (2.97%), and 14 (2.97%) cases were positive, respectively. Only one case was positive for RSV-B, as shown in Table 1 . Although the mean C t values varied from one another, 100% consistency was observed between the multiplex and monoplex real-time RT-PCR assay results for six viruses (Table 1) infection was also investigated in this study (Table 3) . We noted that HPIV-1, -2, -3 were predominantly detected in the age group of AFRS under 5 years. And HPIV-1, -2, RSV-A were more frequently detected among inpatients than outpatients with AFRS. Season distribution was also observed for several human paramyxoviruses. No sex bias was shown. Finally, we compare the performance of our real-time RT-PCR assay with a commercial 2Smart kit for detection of human paramyxoviruses in the clinical setting ( Table 4 ). The data showed that only 36 positives (7.64%) for human paramyxoviruses were detected among 471 clinical specimens from patients with AFRS, which was significantly lower than the results detected by our multiplex real-time RT-PCR assay (in-house), especially for detection of HPIV-1, HPIV-2, and RSV-A. Further analysis indicated that the most discordant results were mainly due to low levels of pathogens with higher C t (36 < C t < 38). We conclude that our multiplex real-time RT-PCR assay showed better performance (higher sensitivity) than the commercial 2Smart kit for detection of human paramyxoviruses in the clinical setting. In conclusion, first, we developed a novel multiplex real-time RT-PCR assay for the rapid detection of six common human paramyxoviruses, which were dominant in patients with AFRS in Qinghai. Second, our method provides a new approach with a higher quality of performance (accuracy, speed, and higher sensitivity) for the detection of common respiratory paramyxoviruses in clinical settings. This two-tube triplex real-time PCR assay provides several advantages. It is more specimen-and time-saving and more cost- Virus (target) N, % Multiplex Mean C t Monoplex Mean C t The authors declare that there are no conflicts of interest. PN and WT conceived and designed the experiments, GL, PN, and RL performed the experiments, GL and WT analyzed the data, and GL and WT wrote the manuscript. Pei-Hua Niu http://orcid.org/0000-0001-5930-4234 Associations between codetected respiratory viruses in children with acute respiratory infections Genetic variability of human metapneumovirus amongst an all ages population in Cambodia between Paramyxoviruses infecting humans: the old, the new and the unknown Respiratory syncytial virus: infection, detection, and new options for prevention and treatment The role of human parainfluenza virus infections in the immunopathology of the respiratory tract Simultaneous detection of respiratory syncytial virus and human metapneumovirus by one-step multiplex real-time RT-PCR in patients with respiratory symptoms The role of multiplex PCR in respiratory tract infections in children Human metapneumovirus infections in hospitalized children Characteristics of human metapneumovirus infection prevailing in hospital wards housing patients with severe disabilities Development of a multiplex RT-PCR for simultaneous diagnosis of human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV) from clinical specimens Acute Respiratory Infections in Children. Disease Control Priorities in Developing Countries New molecular virus detection methods and their clinical value in lower respiratory tract infections in children Real-time PCR in virology Evaluation of a multiplex real-time PCR assay for the detection of respiratory viruses in clinical specimens Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages Two-tube multiplex real-time reverse transcription PCR to detect six human coronaviruses Development and evaluation of a four-tube real-time multiplex PCR assay covering fourteen respiratory viruses, and comparison to its corresponding single target counterparts Development of a multiplex one step RT-PCR that detects eighteen respiratory viruses in clinical specimens and comparison with real-time RT-PCR T A B L E 4 Comparison of performances of our multiplex real-time RT-PCR assay (in-house) and commercial 2Smart kit for detection of human paramyxoviruses in a clinical settingIn-house 2SmartIn-house 2SmartIn-house 2Smart Abbreviations: C t , cycle threshold; HMPV, human metapneumovirus; HPIV, human parainfluenza virus; RSV, respiratory syncytial virus.