Exploiting the high sensitivity of the chemiluminescence phenomenon, an accurate and sensitive point-ofcare test, called the ZstatFlu-II test (ZymeTx, Inc., Oklahoma City, Okla.), was developed to detect influenza virus infections. The ZstatFlu-II test takes 20 min and requires approximately 2 min of "hands-on" time for operational steps. The ZstatFlu-II test does not distinguish between infections with influenza virus types A and B. ZstatFlu-II test results are printed on Polaroid High-Speed Detector Film, allowing test results to be archived. A prototype version of the ZstatFlu-II test was evaluated during the 2000-to-2001 flu season with 300 nasal aspirate specimens from children at a pediatric hospital. Compared to culture, the ZstatFlu-II test had 88% sensitivity and 92% specificity. The Directigen test had a sensitivity of 75% and a specificity of 93%. The sensitivity of the ZstatFlu-II test was significantly higher than that of the Directigen test (P < 0.0574).The annual economic cost of influenza disease in the United States has been estimated at $3 billion to $5 billion (22). The Centers for Disease Control and Prevention reported in 2001 that influenza was associated with about 20,000 deaths nationwide and more than 100,000 hospitalizations (http://www.cdc .gov/ncidod/diseases/flu/fluinfo.htm). Worldwide estimates were considerably higher (16,17,24). Sensitive, specific, and rapid tests for influenza will greatly improve patient health care and reduce costs so that only "flu-positive" patients receive the recently approved antiviral treatments (9, 21). Rapid diagnostics for influenza will also prevent the misuse of antibiotics to "treat" the flu.Influenza disease is caused by influenza virus types A and B. Influenza virus types A and B are Orthomyxoviridae, characterized by the presence of an envelope penetrated by glycoprotein spikes with hemagglutinating and neuraminidase activities. Influenza virus also contains matrix protein, nucleoprotein, and three proteins with polymerase activity and a segmented negative-strand RNA genome (6, 19). These viruses are responsible for winter epidemics of respiratory illness in which the rates of infection are highest among children (5). The shared cardinal sign of fever without localization makes differentiation of influenza from sepsis necessary for proper patient management. Delay in this differentiation could result in unnecessary laboratory testing and treatment for possible bacterial "sepsis" with unnecessary antibiotics (25).Three diagnostic methods for respiratory secretions are in common use. Culture, both shell vial and tube, takes several days. Direct or indirect immunofluorescence assays on exfoliated nasal pharyngeal cells could be done in a few hours but require a high level of expertise (10). Finally, rapid, point-ofcare tests are available to detect the influenza virus (3,12,20,23). All of these tests use an enzyme immunoassay directed at antigens of the viruses (3, 12, 23), with the exception of the ZstatFlu test, which detects influenza virus neur...

We describe the engineering and product development of the chemiluminescent ZstatFlu-II Test kit for influenza diagnostics. The reaction vessel is a chemical implementation device with a polystyrene bottom chamber and a polypropylene top chamber that screw together. The patient's specimen is dispersed in a proprietary diluent and mixed inside the bottom chamber with the influenza viral neuraminidase-specific substrate, 1,2-dioxetane-4,7-dimethoxy-Neu5Ac. Neuraminidase catalysis releases the dioxetane. The top chamber contains 40% NaOH and is sealed at the top with an ABS plastic plug-crush pin assembly. The top chamber floor is 85% thinner at the centre, forming a frangible flap. An automated imaging device serves as an incubator for the chemical implementation devices and also facilitates the piercing of the flap by the crush pin. This action results in NaOH flushing into the bottom chamber, initiating chemiluminescence. The imaging device also exposes the Polaroid high-speed detector film to chemiluminescence. At the end of exposure, the film is automatically processed and ejected. Chemiluminescence from an influenza virus-positive specimen produces a "+"-shaped white image, archiving the diagnostic outcome. The modular ZstatFlu-II test kit components are easily adaptable for the chemiluminescent detection of a wide range of analytes.

The ZstatFlu-II test is a highly sensitive, specific, rapid, point-of-care chemiluminescent diagnostic test for influenza infection. Influenza viral neuraminidase-specific substrate, spiroadamantyl-1,2-dioxetane-4,7-dimethoxy-N-acetyl-neuraminic acid, is at the core of the ZstatFlu-II Test. The enzymatic reaction was carried out at 25 degrees C and neutral pH, representing the optimum assay conditions for influenza types A and B viral neuraminidases. The results were outputted on a Polaroid trade mark High Speed Detector Film. Positive results appeared as a '+'-shaped white film image; negative results produced no image. The 'glow' kinetics, facilitated by a unique combination of light enhancers, also 'tuned' the wavelength of emission to match the spectral properties of the film. The substrate hydrolysed non-enzymatically at acid pH or at temperatures above 25 degrees C. In order to minimize false positives, the ZstatFlu-II Test was formatted with 0.3-0.4 K(m) substrate and freezing the test kit until use. The pH optimization of the ZstatFlu-II test is discussed with reference to model compounds of sialyl-glycosides. A nucleophilic attack or an electrostatic stabilization of a developing carbonium ion under the influence of the adjacent carboxyl group was probably responsible for non-enzymatic hydrolysis of the substrate. Intramolecular general acid catalysis is proposed as a mechanism for the lability of the O-glycosidic linkage of the substrate.