Maiara L Bouzas1, Juliana R Oliveira2, Artur Queiroz3, Kiyoshi F Fukutani4, Aldina Barral5, Annabel Rector6, Elke Wollants6, Els Keyaerts7, Winke Van der Gucht6, Marc Van Ranst7, Kurt Beuselinck8, Camila I de Oliveira9, Johan Van Weyenbergh6, Cristiana M Nascimento-Carvalho10. 1. Postgraduate Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil. Electronic address: maiara.lanna@gmail.com. 2. Postgraduate Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil. 3. Centro de Pesquisas Gonçalo Moniz (CPqGM), Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Brazil. 4. Postgraduate Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil; Department of Biochemistry, Immunology, and Cell Biology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil. 5. Postgraduate Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil; Centro de Pesquisas Gonçalo Moniz (CPqGM), Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Brazil; Department of Pathology, Federal University of Bahia School of Medicine, Salvador, Brazil. 6. Department of Microbiology and Immunology, Laboratory for Clinical and Epidemiological Virology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium. 7. Department of Microbiology and Immunology, Laboratory for Clinical and Epidemiological Virology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium; Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium. 8. Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium. 9. Postgraduate Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil; Centro de Pesquisas Gonçalo Moniz (CPqGM), Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Brazil. 10. Postgraduate Program in Health Sciences, Federal University of Bahia School of Medicine, Salvador, Brazil; Department of Pediatrics, Federal University of Bahia School of Medicine, Salvador, Brazil.
Abstract
BACKGROUND: Virus-specific molecular assays such as real-time polymerase chain reaction (RT-PCR) are regularly used as the gold standard to diagnose viral respiratory tract infections, but simultaneous detection of multiple different pathogens is often challenging. A multiplex digital method of RNA quantification, nCounter (NanoString Technologies), can overcome this disadvantage and identify, in a single reaction, the presence of different respiratory viruses. OBJECTIVES: To evaluate the accuracy of nCounter to identify and quantify RSV-A and RSV-B in nasopharyngeal aspirates (NPA) of children (6-23-months-old) with acute respiratory infection. STUDY DESIGN: NPA was collected at enrolment in a prospective cross-sectional study conducted in Salvador, Brazil. A quantitative RT-PCR with a subgroup-specific primer and probeset for RSV-A and RSV-B was performed in parallel with a customized nCounter probeset containing viral targets in NPA. RESULTS: Of 559 NPA tested, RSV was detected by RT-PCR in 139 (24.9%), by nCounter in 122 (21.8%) and by any method in 158 (28.3%) cases. Compared to the gold standard of qRT-PCR, sensitivity of nCounter was 74.3% (95%CI:63.3%-82.9% RSV-A) and 77.6% (95%CI:66.3%-85.9% RSV-B); specificity was 98.4% (95%CI:96.8%-99.2% RSV-A) and 97.8% (95%CI:96.0%-98.8% RSV-B); positive predictive value was 87.3% (95%CI:76.9%-93.4% RSV-A) and 82.5% (95%CI:71.4%-90.0% RSV-B) and negative predictive value was 96.1% (95%CI:94.1%-97.5% RSV-A), and 96.9% (95%CI:95.1%-98.2% RSV-B). Accuracy was 95.2% (95%CI:93.1%-96.7%) for RSV-A and 95.3% (95%CI:93.3%-96.9%) for RSV-B, while both methods significantly correlated for RSV-A (r = 0.44, p = 8 × 10-5) and RSV-B (r = 0.73, p = 3 × 10-12) quantification. CONCLUSIONS: nCounter is highly accurate in detecting RSV-A/B in NPA. Robustness and high-throughput multiplexing indicate its use in large-scale epidemiological studies.
BACKGROUND: Virus-specific molecular assays such as real-time polymerase chain reaction (RT-PCR) are regularly used as the gold standard to diagnose viral respiratory tract infections, but simultaneous detection of multiple different pathogens is often challenging. A multiplex digital method of RNA quantification, nCounter (NanoString Technologies), can overcome this disadvantage and identify, in a single reaction, the presence of different respiratory viruses. OBJECTIVES: To evaluate the accuracy of nCounter to identify and quantify RSV-A and RSV-B in nasopharyngeal aspirates (NPA) of children (6-23-months-old) with acute respiratory infection. STUDY DESIGN: NPA was collected at enrolment in a prospective cross-sectional study conducted in Salvador, Brazil. A quantitative RT-PCR with a subgroup-specific primer and probeset for RSV-A and RSV-B was performed in parallel with a customized nCounter probeset containing viral targets in NPA. RESULTS: Of 559 NPA tested, RSV was detected by RT-PCR in 139 (24.9%), by nCounter in 122 (21.8%) and by any method in 158 (28.3%) cases. Compared to the gold standard of qRT-PCR, sensitivity of nCounter was 74.3% (95%CI:63.3%-82.9% RSV-A) and 77.6% (95%CI:66.3%-85.9% RSV-B); specificity was 98.4% (95%CI:96.8%-99.2% RSV-A) and 97.8% (95%CI:96.0%-98.8% RSV-B); positive predictive value was 87.3% (95%CI:76.9%-93.4% RSV-A) and 82.5% (95%CI:71.4%-90.0% RSV-B) and negative predictive value was 96.1% (95%CI:94.1%-97.5% RSV-A), and 96.9% (95%CI:95.1%-98.2% RSV-B). Accuracy was 95.2% (95%CI:93.1%-96.7%) for RSV-A and 95.3% (95%CI:93.3%-96.9%) for RSV-B, while both methods significantly correlated for RSV-A (r = 0.44, p = 8 × 10-5) and RSV-B (r = 0.73, p = 3 × 10-12) quantification. CONCLUSIONS: nCounter is highly accurate in detecting RSV-A/B in NPA. Robustness and high-throughput multiplexing indicate its use in large-scale epidemiological studies.
Authors: Kiyoshi F Fukutani; Cristiana M Nascimento-Carvalho; Maiara L Bouzas; Juliana R Oliveira; Aldina Barral; Tim Dierckx; Ricardo Khouri; Helder I Nakaya; Bruno B Andrade; Johan Van Weyenbergh; Camila I de Oliveira Journal: Front Microbiol Date: 2018-11-09 Impact factor: 5.640