Real-Time Detection of Influenza A, Influenza B, and Respiratory Syncytial Virus A and B in Respiratory Specimens by Use of Nanoparticle Probes

Abstract

Seasonal epidemics of influenza and respiratory syncytial virus are responsible for significant morbidity and mortality worldwide. Infrequently, novel or reemergent strains of influenza A virus have caused rapid, severe global pandemics resulting in millions of fatalities. The ability to efficiently and accurately detect and differentiate respiratory viruses is paramount for effective treatment, infection control, and epidemiological surveillance. We evaluated the ability of two FDA-cleared nucleic acid-based tests, the semiautomated respiratory virus nucleic acid test (VRNAT) and the fully automated respiratory virus nucleic acid test SP (RVNAT SP ) (Nanosphere Inc., Northbrook, IL) to detect influenza A virus, influenza B virus, and respiratory syncytial virus A and B (RSV A/B) from clinical nasopharyngeal swab specimens. Detection of viral RNA in both tests is based on nucleic acid amplification followed by hybridization to capture probes immobilized on a glass slide. A novel technology utilizing gold nanoparticle-conjugated probes is utilized to detect the presence of captured target DNA. This microarray-based approach to detection has proven to be more sensitive than the traditional culture/direct fluorescent-antibody assay (DFA) method for detecting RSV and influenza viruses in clinical specimens, including the novel 2009 H1N1 strain. Specifically, we report 98.0% sensitivity and 96.5% specificity for the VRNAT compared to culture/DFA. Further, the VRNAT detected virus in an additional 58% of specimens that were culture negative. These data were confirmed using bidirectional sequencing. Evaluation of the fully automated RVNAT SP , which is built on the same detection technology as the VRNAT but contains an updated processor enabling complete automation, revealed the two tests to be functionally equivalent. Thus, the RVNAT SP is a fully automated sample-to-result test capable of reliable detection of select respiratory viruses directly from clinical specimens in 3.5 h.Influenza virus and respiratory syncytial virus (RSV) infect millions of individuals annually. Influenza virus is typically associated with infections of the upper respiratory tract and can cause mild to severe illness, with symptoms including abrupt onset of fever, malaise, severe myalgia, and a nonproductive cough. The bulk of illness is attributed seasonal epidemics of influenza virus types A and B, while type C is far less prevalent and rarely causes severe disease (9, 23). Type A influenza virus is often associated with more-severe symptoms, morbidity, and mortality owing to a greater genetic diversity typified by chromosomal reassortment between human and avian viruses. Such genetic reassortments are responsible for the emergence of novel subtypes of influenza A virus, including the infamous H1N1 Spanish flu virus of 1918 and the more recent 2009 H1N1 or "swine flu" virus (26).RSV is capable of causing severe symptoms in infants, young children, and immunocompromised individuals (9,24,28) and is the leading cause of hospitalization in ch...

Molecular diagnosis of respiratory virus infections

“…Although earlier developmental papers describe the analytical performance of the Clart ® PneumoVir assay, in the absence of comparative studies with large numbers of clinical specimens, the clinical performance remains largely unknown 139 . Another commercially available real time RT-PCR that uses nanoparticles for the detection of four respiratory viruses, including RSV, has recently been FDA-cleared 142 . There are two versions of this test: the manual VNAT test and the fully automated RVNAT SP version that both detect four respiratory viruses, including influenza A and B and RSV A and B, by multiplex PCR followed by hybridization to probes immobilized on glass slides.…”

“…The test uses gold nanoparticle-labeled probes to detect captured target DNA. In a two-site evaluation involving 720 NP specimens, including 49 RSV positives where culture and DFA were used as the reference standard, the RVNAT SP test had a sensitivity and specificity of 91.7% and 98.4%, respectively, for RSV 142 . Performance studies involving comparisons of two or more of these commercial multiplex assays will be required to determine the "true" performance of these assays; these comparisons are eagerly awaited.…”

Evaluation of Alere i Influenza A&B for Rapid Detection of Influenza Viruses A and B

“…As a result, specimens with negative RIDTs are usually tested subsequently by more sensitive culture or molecular assays. PCR-based molecular assays have shown excellent clinical utility for the detection and identification of influenza viruses; numerous FDA-cleared commercial devices are now available (18,23,24).…”

Emerging Technologies for the Clinical Microbiology Laboratory

“…Clinical evaluations have reported sensitivities of 96.6% to 100% for influenza virus A, 96.8% to 100% for influenza virus B, and 89.8% to 91.7% for RSV, with specificities of Ͼ96.5% for all targets (100,103,104), which in one comparative study was superior to results for a traditional RT-PCR test (103).…”

“…4) has offers microarraybased tests for identification of respiratory viruses (RVϩ), C. difficile (CDF), blood cultures containing Gram-positive bacteria (BC-GP) or Gram-negative bacteria (BC-GN), and identification of genetic variants, including Factor V Leiden and CYP450 2C19 *2 and *3, which impact patients with coagulation disorders (94)(95)(96)(97)(98)(99)(100)(101)(102).…”