Diagnostic accuracy of digital RNA quantification versus real-time PCR for the detection of respiratory syncytial virus in nasopharyngeal aspirates from children with acute respiratory infection

Bouzas, Maiara L.; Oliveira, Juliana R.; Queiroz, Artur T. L.; Fukutani, Kiyoshi F.; Barral, Aldina; Rector, Annabel; Keyaerts, Els; Gucht, Winke Van der; Ranst, Marc Van; Beuselinck, Kurt; Oliveira, Camila I. de; Weyenbergh, Johan Van; Nascimento‐Carvalho, Cristiana M.; Infection, Acute Respiratory

doi:10.1016/j.jcv.2018.07.003

Cited by 11 publications

(8 citation statements)

References 17 publications

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“…Due to financial limitations, only the pathogens reliably detected in both stages were used in subsequent experiments. Nonetheless, we have confirmed the detection of RSV-A/RSV-B, as shown here by nCounter, using RT-PCR ( Bouzas et al, 2018 ), and of H. influenzae, M. catarrhalis, S. aureus , and S. pneumoniae by 16S next-generation sequencing ( Andrade et al, 2016 ).…”

Section: Methodssupporting

confidence: 79%

“…As described before in Fukutani et al (2015) and Bouzas et al (2018) , the nCounter probe set used in this study was extensively tested and optimized for semihigh throughput analysis. First, sensitivity, linearity, and absence of cross-reaction among the respiratory bacterial and viral targets were tested with a panel of positive controls obtained from the collection of the Laboratory of Clinical Virology (KU Leuven).…”

Section: Methodsmentioning

confidence: 99%

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In situ Immune Signatures and Microbial Load at the Nasopharyngeal Interface in Children With Acute Respiratory Infection

et al. 2018

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Acute respiratory infection (ARI) is the most frequent cause for hospitalization in infants and young children. Using multiplexed nCounter technology to digitally quantify 600 human mRNAs in parallel with 14 virus- and 5 bacterium-specific RNAs, we characterized viral and bacterial presence in nasopharyngeal aspirates (NPA) of 58 children with ARI and determined the corresponding in situ immune profiles. NPA contained different groups of organisms and these were classified into bacterial (n = 27), viral (n = 5), codetection [containing both viral and bacterial transcripts (n = 21), or indeterminate intermediate where microbial load is below threshold (n = 5)]. We then identified differentially expressed immune transcripts (DEITs) comparing NPAs from symptomatic children vs. healthy controls, and comparing children presenting NPAs with detectable microbial load vs. indeterminate. We observed a strong innate immune response in NPAs, due to the presence of evolutionarily conserved type I Interferon (IFN)-stimulated genes (ISG), which was correlated with total bacterial and/or viral load. In comparison with indeterminate NPAs, adaptive immunity transcripts discriminated among viral, bacterial, and codetected microbial profiles. In viral NPAs, B cell transcripts were significantly enriched among DEITs, while only type III IFN was correlated with viral load. In bacterial NPAs, myeloid cells and coinhibitory transcripts were enriched and significantly correlated with bacterial load. In conclusion, digital nCounter transcriptomics provide a microbial and immunological in situ “snapshot” of the nasopharyngeal interface in children with ARI. This enabled discrimination among viral, bacterial, codetection, and indeterminate transcripts in the samples using non-invasive sampling.

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Section: Methodssupporting

confidence: 79%

Section: Methodsmentioning

confidence: 99%

In situ Immune Signatures and Microbial Load at the Nasopharyngeal Interface in Children With Acute Respiratory Infection

et al. 2018

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“…Standards and negative controls were included and tested with each PCR assay. One-step RT-PCR (Qiagen QuantiTect) targeting the conserved region of the F gene was performed using described primers and probes [17,18]. RSV quantification is reported as log 10 copies/mL.…”

Section: Rsv Loadsmentioning

confidence: 99%

Viral Load Dynamics and Clinical Disease Severity in Infants With Respiratory Syncytial Virus Infection

Garcia-Maurino

Moore‐Clingenpeel

Thomas

et al. 2018

The Journal of Infectious Diseases

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Background. The association between respiratory syncytial virus (RSV) loads and clinical outcomes in children remains to be defined. In most studies, viral loads (VL) were evaluated in hospitalized children and at a single time-point. We investigated the relationship between VLs and disease severity in both outpatients and inpatients with RSV infection.Methods. We enrolled previously healthy children with RSV infection. Disease severity was defined by level of care (outpatients vs ward vs pediatric intensive care unit [PICU]), and a clinical disease severity score (CDSS). Nasopharyngeal VLs by polymerase chain reaction and CDSS were measured at enrollment and daily in inpatients. VL decay according to disease severity was analyzed using linear mixed modeling.Results. From February 2015 to March 2017, we enrolled 150 infants: 39 outpatients and 111 inpatients. VLs were higher in outpatients than in age-matched inpatients. Among inpatients, initial VLs were comparable in ward and PICU patients, and preceded the peak CDSS. However, after excluding infants treated with steroids, those hospitalized in the ward had higher VLs than infants requiring PICU care (P < .001). Dynamic analyses showed that VL decay was delayed in PICU patients, especially in those treated with steroids.Conclusions. Higher VLs at presentation and a faster and consistent VL decline were both associated with less severe RSV disease in children.

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“…Neena Kanwar et al compared the diagnostic accuracy of the two DIA tests with the real-time RT-PCR assay results as a reference, showing a sensitivity of 71.15% for BD VeritorTM System and 80.77% for Quidel ® Sofia ® RSV FIA system, and the specificity of 100% for both systems (15). The sensitivity and specificity of the nCounter (NanoString Technologies), which is a multiplex digital method of RNA quantification, in comparison to real-time RT-PCR were found to be 74.3% and 98.4% for RSV-A, 77.6% and 97.8% for RSV-B, respectively (16). Compared with the results of those studies, the colloidal-gold-Genesis showed higher sensitivity and lower specificity with a higher Youden index.…”

Section: Discussionmentioning

confidence: 99%

Colloidal Gold Assay, a New Screening Test for Detection of RSV Infection: A Controlled Trial

Liu

Tao

et al. 2019

Iran J Pediatr

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Background: Respiratory syncytial virus (RVS) is the main cause of morbidity and mortality in the pediatric population. Objectives: The objective of the study was to compare the diagnostic accuracies of the colloidal-gold-Genesis and newly developed real-time RT-PCR-IngeniGen with an admitted real-time RT-PCR-HuaRuiAn as the gold standard for respiratory syncytial virus detection. Methods: These three assays were performed on collected specimens. Results: Altogether 258 specimens were studied. Among them, real-time RT-PCR-HuaRuiAn detected the respiratory syncytial virus in 166 samples, while colloidal-gold-Genesis detected 171 samples and real-time RT-PCR-IngeniGen detected 168 samples as positive. The overall sensitivity for the detection of the respiratory syncytial virus in comparison to real-time RT-PCR-HuaRuiAn was 95.2% for colloidal-gold-Genesis and 100% for real-time RT-PCR-IngeniGen. The specificity was 85.9% for colloidal-gold-Genesis and 97.8% for real-time RT-PCR-IngeniGen. The positive likelihood ratio was 6.75 for colloidal-gold-Genesis and 45.45 for real-time RT-PCR-IngeniGen. The negative likelihood ratio was 0.06 for colloidal-gold-Genesis and 0.00 for real-time RT-PCR-IngeniGen. The cycle threshold value of real-time RT-PCR-IngeniGen was higher than that of real-time RT-PCR-HuaRuiAn. Conclusions: Colloidal-gold-Genesis is a good alternative for fast screening of respiratory syncytial virus detection.

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Diagnostic accuracy of digital RNA quantification versus real-time PCR for the detection of respiratory syncytial virus in nasopharyngeal aspirates from children with acute respiratory infection

Cited by 11 publications

References 17 publications

In situ Immune Signatures and Microbial Load at the Nasopharyngeal Interface in Children With Acute Respiratory Infection

In situ Immune Signatures and Microbial Load at the Nasopharyngeal Interface in Children With Acute Respiratory Infection

Viral Load Dynamics and Clinical Disease Severity in Infants With Respiratory Syncytial Virus Infection

Colloidal Gold Assay, a New Screening Test for Detection of RSV Infection: A Controlled Trial

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