title: Microfluidic chip for molecular amplification of influenza A RNA in human respiratory specimens.
abstract: A rapid, low cost, accurate point-of-care (POC) device to detect influenza virus is needed for effective treatment and control of both seasonal and pandemic strains. We developed a single-use microfluidic chip that integrates solid phase extraction (SPE) and molecular amplification via a reverse transcription polymerase chain reaction (RT-PCR) to amplify influenza virus type A RNA. We demonstrated the ability of the chip to amplify influenza A RNA in human nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens collected at two clinical sites from 2008-2010. The microfluidic test was dramatically more sensitive than two currently used rapid immunoassays and had high specificity that was essentially equivalent to the rapid assays and direct fluorescent antigen (DFA) testing. We report 96% (CI 89%,99%) sensitivity and 100% (CI 95%,100%) specificity compared to conventional (bench top) RT-PCR based on the testing of n = 146 specimens (positive predictive value = 100%(CI 94%,100%) and negative predictive value = 96%(CI 88%,98%)). These results compare well with DFA performed on samples taken during the same time period (98% (CI 91%,100%) sensitivity and 96%(CI 86%,99%) specificity compared to our gold standard testing). Rapid immunoassay tests on samples taken during the enrollment period were less reliable (49%(CI 38%,61%) sensitivity and 98%(CI 98%,100%) specificity). The microfluidic test extracted and amplified influenza A RNA directly from clinical specimens with viral loads down to 10³ copies/ml in 3 h or less. The new test represents a major improvement over viral culture in terms of turn around time, over rapid immunoassay tests in terms of sensitivity, and over bench top RT-PCR and DFA in terms of ease of use and portability.
authors.affiliation: Department of Mechanical Engineering, Boston University, Boston, Massachusetts, United States of America.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Cao
authors.forename: Madhumita
authors.initials: M
authors.lastname: Mahalanabis
authors.forename: Jessie
authors.initials: J
authors.lastname: Chang
authors.forename: Brendan
authors.initials: B
authors.lastname: Carey
authors.forename: Christopher
authors.initials: C
authors.lastname: Hsieh
authors.forename: Ahjegannie
authors.initials: A
authors.lastname: Stanley
authors.forename: Christine A
authors.initials: CA
authors.lastname: Odell
authors.forename: Patricia
authors.initials: P
authors.lastname: Mitchell
authors.forename: James
authors.initials: J
authors.lastname: Feldman
authors.forename: Nira R
authors.initials: NR
authors.lastname: Pollock
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Klapperich
mesh_terms: D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009980:Influenza A virus / Q000235:genetics*; D007251:Influenza, Human / Q000175:diagnosis / Q000821:virology; D044085:Microfluidics / Q000295:instrumentation*; D008969:Molecular Sequence Data; D009305:Nasopharynx / Q000821:virology*; D019095:Point-of-Care Systems; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid
chemical_list: D012367:RNA, Viral