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The performance of a new rapid lateral-flow chromatographic membrane immunoassay test kit for detection of influenza virus was evaluated and compared to that of viral culture in respiratory secretions collected from 400 adults and children seen at three large university hospitals during the recent 2003 influenza season. The rapid test provided results in 15 min, with excellent overall performance statistics (sensitivity, 94.4%; specificity, 100%; positive predictive value, 100%; negative predictive value, 97.5%). Both influenza A and B type viruses were reliably detected, with no significant difference in performance statistics noted by influenza virus type or by the center performing the test.
Influenza virus is a major cause of respiratory infection in both adults and children and is a common cause of hospitalization, especially in young children, elderly adults, and persons with chronic diseases (17, 20). Influenza epidemics also account for over 47,000 deaths annually in the United States. Furthermore, three global pandemics during the 20th century caused over 50 million deaths (22). Readily available, rapid, and accurate detection methods for influenza virus allow for prompt administration of appropriate antiviral therapy and judicious use of antibiotics, assist in isolation of patients in hospitals and emergency centers to reduce health care-associated spread of infection, and identify local epidemics of influenza in a timely manner (10, 13-15, 19, 21, 22). Rapid detection of influenza virus also is currently important because of increased concern for pandemic influenza caused by either naturally occurring strains, such as avian H5N1, or altered strains that may be used in an act of bioterrorism (7, 8, 18). Also, the diagnosis of influenza based on clinical grounds alone may be inaccurate, because the presenting symptoms of influenza are similar to those caused by other infectious agents (13). Furthermore, the rapid and accurate determination that a severe respiratory or flu-like illness is caused by influenza virus rather than severe acute respiratory syndrome-associated coronavirus or a bioterrorism agent, such as smallpox or tularemia, is helpful not only to the individual patient but also from the public health perspective (6).
Currently, there are at least seven different test kits approved by the Food and Drug Administration for detection of influenza virus in respiratory samples (2-4, 9, 12, 16, 21, 23). However, not all available methods distinguish the type of influenza virus present in the sample, and those that do distinguish type A and B influenza viruses have not shown consistently reliable performance for both types of virus (3, 4, 9, 12). This recent multicenter study documented promising performance of a new rapid lateral-flow chromatographic immunoassay for both detection and differentiation of influenza A and B type viruses in respiratory samples.
(This study was presented, in part, at the Pediatric Academic Societies Annual Meeting, 1 to 4 May 2004, in San Francisco, Calif., and at the 20th Annual Clinical Virology Symposium, 25 to 28 April 2004, Clearwater, Fla.)
The tests results obtained with a new rapid lateral-flow chromatographic membrane immunoassay (Xpect FluA/B; Remel Inc., Lenexa, Kans.) were compared to those with the reference standard of viral culture in 400 respiratory specimens collected from children and adults who presented between January and April 2003 with respiratory or flu-like symptoms to one of three hospitals in three geographically distinct areas: Texas Children's Hospital, Houston, Tex. (n = 166), University of South Florida Affiliated Hospitals and DSI Reference Laboratory, Fort Myers, Fla. (n = 151), and SUNY Upstate Medical University Affiliated Hospitals, New York, N.Y. (n = 83). Most (239 of 400, or 59.75%) specimens were nasal washes, 122 of 400 (30.5%) were nasopharyngeal swabs, 30 of 400 (7.5%) were throat swabs, 4 of 400 (1%) were tracheal aspirates, 3 of 400 (0.75%) were sputum, and 2 of 400 (0.5%) were obtained by bronchoalveolar lavage. Both fresh samples and samples cryopreserved for less than 3 months were analyzed at each institution.
Virology technicians or technologists at all centers received instruction on the new test procedures and were required to pass (>90%) a blinded proficiency test on six coded samples, administered daily for 4 days, prior to starting testing on clinical samples included in this study. Rapid tests were performed according to the manufacturer's instructions during weekday, day shift hours. Briefly, the test detects influenza virus antigen in a test device that contains a sample well connected to reading wells that contain separate membrane strips for influenza A and influenza B viruses (Fig. (Fig.1).1). Each sample was mixed with a specimen diluent that contained buffered saline, detergent, a mucolytic agent, and preservative. Then, 0.20 ml was transferred by pipette into the middle of the test well of the device. A positive test was indicated by two black bands in the reading well, one in the test (T) region and one in the control (C) region. A negative test was indicated by only one black band in the C region. The absence of any black bands in the T or C regions represented an invalid test. Test readings were performed and recorded after 15 and 30 min of incubation. Quality control procedures were performed and recorded for each test run or 24-h period and included both Flu A+/Flu B− and Flu A−/Flu B+ controls provided by the test kit, as well as in-house positive and negative controls for each virus. All specimens were also inoculated that same day into cell culture monolayers of human foreskin fibroblast, human lung carcinoma (A549), human epithelial (HEp2), and rhesus monkey kidney (RhMK) cells and examined daily for cytopathic effect using light microscopy. Hemadsorption with a 0.4% suspension of guinea pig red blood cells was performed on days 2, 5, and 14 of incubation of RhMK cell cultures. Virus identification was confirmed by an immunofluorescence assay with type-specific antibodies. At one institution (Texas Children's Hospital), all picornaviruses were discriminated by acid lability testing to distinguish between rhinoviruses and enteroviruses. Samples with discrepant results between viral culture and the rapid influenza virus test were cryopreserved and analyzed by reverse transcription-PCR (RT-PCR) using primers able to detect and differentiate influenza A and B viruses (1).
Viral cultures positive for influenza virus, type A or B, were considered true positives. Sensitivity, specificity, and positive and negative predictive values were calculated using two-by-two contingency tables. Differences between tests were analyzed using Fisher's exact test. Because rapid testing for influenza virus may be performed to screen persons during a pandemic or other event affecting large numbers of people, confidence intervals for proportions were calculated, to estimate with 95% confidence, the intervals that contain the sensitivity, specificity, and predictive values for the general population, estimated in this analysis to be 1,000,000. Using the confidence intervals, one can generalize results from the sample size of 400 to a large population, and the estimation predicts the interval that will contain the rapid assay's performance. The importance of this test performance seems especially relevant because contemporary concerns about global pandemics of influenza and severe acute respiratory syndrome-associated coronavirus, as well as the possibility of a biological warfare event, suggest it may some day be necessary to screen large numbers of persons with febrile respiratory illness of undetermined etiology.
Of the 400 specimens in this study, 207 (51.75%) had a negative viral culture and 193 (48.25%) grew at least one virus (Table (Table1).1). Dual viral infections were detected in four specimens (one with influenza A virus and adenovirus, one with influenza A virus and respiratory syncytial virus, one with influenza B virus and cytomegalovirus, and one with picornavirus and adenovirus), to give a total of 197 viruses isolated during the study period. The mean duration of time that elapsed until viral cultures were detected as positive was 4.43 ± 2.87 days, and 87.6% of cultures were positive within 7 days.
The overall sensitivity of the rapid test to detect both types of influenza virus was slightly higher (95.2 versus 94.4%) at the 30-min reading than the 15-min reading, but specificity and predictive values were essentially the same at both readings. Furthermore, no significant differences were observed in the ability of this new rapid test to detect influenza A or B virus, and the results were generalized with 95% confidence to a population of at least 1,000,000 (Table (Table2).2). Also, no significant differences were found when performance statistics were analyzed by center site.
There were no false-positive tests observed at either the 15- or 30-min incubation time for the rapid test. However, there were seven false-negative results at 15 min and six false-negative results at the 30-min incubation time. Only nasal washes (three) and nasopharyngeal swabs (three) had false-negative results. Overall, the specificity of the rapid assay was 100% for all specimen types. Sensitivity was 100% in throat swabs, 96.1% in nasal washes, and 87.9% nasopharyngeal swabs.
False-negative results were detected at all three centers, and testing by RT-PCR of the five specimens with apparent discrepant results confirmed four of the false-negative test results (Table (Table33).
The new lateral-flow chromatographic membrane immunoassay evaluated in this study was both highly sensitive and specific in detecting and differentiating influenza A and B viruses in respiratory specimens collected from patients in three different geographic locations during a recent influenza season in the United States. Furthermore, the results appeared favorable when statistically generalized to a larger population, making this assay potentially useful for screening large numbers of individuals. Many previous studies on the performance of rapid assays for detecting influenza virus were limited because they were conducted at a single center or during an influenza season where only type A predominated (5, 13, 14, 16, 22). In addition, in the few studies in which influenza B virus circulated, significant rates of false-negative tests have been observed (2, 3, 4, 12). Of interest, dual infections with one type of influenza virus and another virus were observed in this study, providing a reminder that a positive rapid test for influenza A or B virus does not eliminate the possibility that the patient may be coinfected with another virus that may be contributing to their symptoms.
The current availability of at least seven different test kits for the rapid detection of influenza virus in clinical samples not only enhances individual patient care, but also may help control the spread of influenza if infected individuals are accurately diagnosed and treated promptly and if outbreaks are identified early and controlled by timely immunization practices (5, 11, 16, 17). However, healthcare workers caring directly for patients, as well as laboratory directors and public health officials, should be aware of the performance characteristics, availability, cost, and reimbursement issues associated with each rapid test and choose the best one for their specific needs and, once implemented, monitor the performance of the test in their particular setting.
This study was supported, in part, by Remel, Inc.