Development of a real-time multiplex RSV detection assay for difficult respiratory samples, using ultrasone waves and MNAzyme technology

Background: Elderly infected with Human Respiratory Syncytial Virus (RSV) often bear low viral loads that stay below the detection limits of commercial assays. A more sensitive detection of RSV infections can improve patient management, guide containment strategies, and possibly prevent morbidity and mortality among populations most severely affected by RSV. Objective: To test the sensitivity for RSV detection by using an alternative extraction method in combination with a new amplification procedure. Study design: Nasopharyngeal washes and sputum samples (n = 78) form clinical cases, and broncheo-alveolar lavages (n = 27) from an experimental RSV rat model were obtained. An ultrasone-based RNA extraction method was combined with a multi-component Nucleic Acid enzymes (MNAzyme) amplification procedure for simultaneous detection of RSV-A, RSV-B, and an Internal Extraction control IEC. Results: Compared to standard real-time PCR technology, this method resulted in an increased detection sensitivity, ranging from 0.9 to 4.93 log (average 2.05 +/- 1.01) for RSV-A and 0.76 to 4.28 log (average 1.30 +/- 0.92) for RSV-B. Conclusions: An ultrasone-based extraction method with MNAzyme amplification resulted in improved detection of RSV in different respiratory samples, including sputum. This generic method for nucleic acid extraction should be readily applicable for any other respiratory pathogen. (C) 2009 Elsevier B.V. All rights reserved.