Clinical evaluation of a panel of multiplex quantitative real-time reverse transcription polymerase chain reaction assays for the detection of 16 respiratory viruses associated with community-acquired pneumonia

Zhang, Dan; Mao, Haiyan; Fang, Zhiguo; Pan, Junhang; Yan, Huan; Tang, Hongfeng; Yan, Shu; Yun, Zhao; Cheng, Xiaoli; Hong, Tao; Zhang, Yanjun

doi:10.1007/s00705-018-3921-8

Cited by 12 publications

(11 citation statements)

References 27 publications

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“…Nucleic acids were extracted from 200-µL samples using the cador Pathogen 96 QIAcube HT Kit (Qiagen) for automated viral DNA and RNA extraction with the QIAcube HT System. The samples were tested individually by qRT-PCR using specific primers and probes targeting different genomes according to our previously reported qRT-PCR methods [18]. The qRT-PCR assays were used to detect 16 different respiratory viruses: CoV (229E, NL63, OC43 and HKU1), PIV1-4, IV types A and B, RSV types A and B, HRV/EV, HMPV, human adenovirus (ADV), and human bocavirus (HBoV).…”

Section: Virologic Methodsmentioning

confidence: 99%

Respiratory virus associated with surgery in children patients

et al. 2019

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Background Viral respiratory infection (VRI) is a common contraindication to elective surgery. Asymptomatic shedding among pediatric surgery patients (PSPs) could potentially lead to progression of symptomatic diseases and cause outbreaks of respiratory diseases. The aim of this study is to investigate the incidence of infection among mild symptomatic PSP group and asymptomatic PSP group after surgical procedure. Methods We collected the induced sputum from enrolled 1629 children (under 18 years of age) with no respiratory symptom prior to pediatric surgery between March 2017 and February 2019. We tested 16 different respiratory virus infections in post-surgery mild symptomatic PSP group and asymptomatic PSP group using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assay panel. We analyzed symptom data and quantitative viral load to investigate the association between viruses, symptoms and viral quantity in qRT-PCR-positive PSPs. Results Out of 1629 children enrolled, a total of 204 respiratory viruses were present in 171 (10.50%) PSPs including 47 patients with mild symptoms and 124 with no symptoms after surgery. Commonly detected viruses were human rhino/enterovirus (HRV/EV, 42.19%), parainfluenza virus 3 (PIV3, 24.48%), coronavirus (CoV NL63, OC43, HKU1, 11.46%), and respiratory syncytial virus (RSV, 9.9%). PIV3 infection with a higher viral load was frequently found in PSPs presenting with mild symptoms, progressing to pneumonia with radiographic evidence after surgery. HRV/EV were the most commonly detected pathogens in both asymptomatic and mild symptomatic PSPs. CoV (OC43, HKU1) infections with a higher viral load were mostly observed in asymptomatic PSPs progressing to alveolar or interstitial infiltration. Conclusions Our study suggested that PIV3 is a new risk factor for VRI in PSPs. Employing a more comprehensive, sensitive and quantitative method should be considered for preoperative testing of respiratory viruses in order to guide optimal surgical timing.

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Section: Virologic Methodsmentioning

confidence: 99%

Respiratory virus associated with surgery in children patients

et al. 2019

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“…Before pooling, we used the cador Pathogen 96 QIAcube HT Kit (Qiagen) for automated viral DNA and RNA extraction with the QIAcube HT System. The presence of each virus was tested by individual qRT-PCR using specific primers and probes targeting different genomes [23]. The Ct values for the ADV, Flu A, PIV3, OC43, and HMPV were 23.9, 26.5, 17.1, 29.2, and 27.1, respectively.…”

Section: Clinical Virus Specimenmentioning

confidence: 99%

“…Following nucleic acid extraction by the four kits (Table 5), their individual performance with regard to the yield of viral nucleic acids was compared using qRT-PCR [23]. Specific qRT-PCR protocols were individually performed in duplicate (n = 16) for each virus and each extract (n = 8), using the 7500 Real Time PCR System (Applied Biosystems) for quantification of the ADV, OC43, Flu A, HMPV, and PIV3.…”

Section: Molecular Confirmation Of Viral Infectionmentioning

confidence: 99%

Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples

et al. 2018

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BackgroundNumerous protocols for viral enrichment and genome amplification have been created. However, the direct identification of viral genomes from clinical specimens using next-generation sequencing (NGS) still has its challenges. As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS, it is still valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly.ResultsIn this study, we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits: QIAamp Viral RNA Mini Kit (VRMK), QIAamp MinElute Virus Spin Kit (MVSK), RNeasy Mini Kit (RMK), and RNeasy Plus Micro Kit (RPMK), using a mixed respiratory clinical sample without any pre-treatment. This sample contained an adenovirus (ADV), influenza virus A (Flu A), human parainfluenza virus 3 (PIV3), human coronavirus OC43 (OC43), and human metapneumovirus (HMPV). The quantity and quality of the viral extracts were significantly different among these kits. The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK. The MVSK had the lowest Ct values for ADV and PIV3. The RMK revealed the lowest detectability for HMPV and PIV3. The most effective rate of NGS data at 67.47% was observed with the RPMK. The other three kits ranged between 12.1–26.79% effectiveness rates for the NGS data. Most importantly, compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK. The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV, while the RMK revealed the best extraction efficiency by NGS analysis.ConclusionsThe evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis. The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples. In addition, viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus. Our results highlighted the importance of nucleic acid extraction kit selection, which has a major impact on the yield and number of viral reads by NGS analysis. Therefore, the choice of extraction method for a given viral pathogen needs to be carefully considered.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5152-5) contains supplementary material, which is available to authorized users.

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“…Laboratory-developed and commercial multiplex real-time PCR assays, which detect a large number of different pathogens, have been developed and implemented for routine diagnostic application. These assays are highly sensitive and specific (7)(8)(9)(10)(11). However, they are complex to perform, involving several testing steps, including nucleic acid extraction prior to amplification and analysis, and require a laboratory turnaround time (TAT) of five to six hours.…”

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confidence: 99%

Impact of Rapid Molecular Detection of Respiratory Viruses on Clinical Outcomes and Patient Management

Kuypers

2019

J Clin Microbiol

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To determine if rapid molecular testing for respiratory viruses in patients with respiratory illnesses can provide advantages to patients and hospitals, rigorous investigations on the impacts of using these assays are required. Well-conducted studies are needed to inform decisions about implementation of new rapid assays to replace standard molecular testing or to initiate testing in laboratories that are currently not doing molecular tests for respiratory viruses due to the complex nature of standard panels. In this issue of the Journal of Clinical Microbiology, N. Wabe et al. (J Clin Microbiol 57:e01727-18, 2019, https://doi.org/10.1128/JCM.01727-18) report the results of their evaluation of the impact of using a rapid molecular test for influenza A/influenza B and RSV on outcomes for adults hospitalized with respiratory illness. The median time from admission to test result of the rapid test was 7.5 h compared to 40.3 h for the standard PCR assay. Compared to the use of the standard molecular assay, use of a rapid test significantly shortened time in the hospital and reduced the number of other microbiology tests performed. The authors concluded that rapid PCR testing of adults hospitalized with respiratory illnesses could provide benefits to both the patients and the hospital. Patients were able to leave the hospital earlier and a greater proportion of them had received their test results before discharge, which would allow appropriate treatment to be provided more quickly.

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Clinical evaluation of a panel of multiplex quantitative real-time reverse transcription polymerase chain reaction assays for the detection of 16 respiratory viruses associated with community-acquired pneumonia

Cited by 12 publications

References 27 publications

Respiratory virus associated with surgery in children patients

Respiratory virus associated with surgery in children patients

Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples

Impact of Rapid Molecular Detection of Respiratory Viruses on Clinical Outcomes and Patient Management

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