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...
The term multiplex PCR refers to simultaneous amplification of more than one target in a single PCR. This method has some advantages but presents the possibility of competition between multiple targets for a finite number of reagents, which may invalidate the assay.The Roche Cobas AMPLICOR multiplex PCR for Chlamydia trachomatis and Neisseria gonorrhoeae, a U.S. Food and Drug Administration-approved assay, can detect both C. trachomatis and N. gonorrhoeae from a single specimen. PCR amplification of C. trachomatis and N. gonorrhoeae proceeds in one tube with a shared enzyme and shared nucleotides but independent biotinylated primers. An optional internal control (IC) permits detection of amplification inhibition. The IC DNA has primer binding sequences identical to those of the C. trachomatis target. Detection is accomplished by using oligonucleotide probes that are unique for C. trachomatis, N. gonorrhoeae, and IC, respectively, with colorimetric quantification by spectophotometer. A negative assay result is valid if the IC optical density (OD) is Ն0.2, indicating successful amplification (Method Manual, Cobas Amplicor, Roche Diagnostics, 12/1999, Revision 3.0).We performed 580 multiplex PCRs on endocervical and urethral swab specimens, and 58 assays yielded positive results for C. trachomatis alone (OD Ն 2.0), 13 assays yielded positive results for N. gonorrhoeae alone (OD Ն 3.5), and 4 assays yielded positive results for both. Of the 58 assays positive for C. trachomatis, 13 (22.4%) had IC OD values that were Ͻ0.2 (mean OD, 0.059). The results of these 13 assays were correctly interpreted as C. trachomatis positive (mean C. trachomatis OD, 3.187). Because of the failure of the IC to amplify, the N. gonorrhoeae OD values for these 13 were invalid. The limiting reagent could be the primer, shared by C. trachomatis and IC. In the absence of IC amplification, it was not clear that there were a sufficient number of reagents, aside from the primer, to amplify N. gonorrhoeae. Of these 13 assays, 11 had OD values interpreted as negative for N. gonorrhoeae (mean N. gonorrhoeae OD, 0.047). Five of these specimens from the 11 assays were cultured and yielded negative results. Two additional specimens that turned out to be N. gonorrhoeae culture positive had equivocal N. gonorrhoeae OD values of 2.013 and 3.492, respectively, resolvable as positive by duplicate repeat testing.A Roche Molecular Systems study suggested that competitive inhibition occurs when the relative concentration of one target is extremely high and that the competition is for reagents other than the primer. This Roche paper discusses the optional use of the IC for increased sensitivity as well as retesting of existing specimens to eliminate nonspecific, labile polymerase inhibition, which we saw in five specimens not discussed here (1). However, we had a very high rate of competitive inhibition not correctable by repetition. Experimental dilution of the specimens did result in IC amplification, but also, in one case, converted a positive C. trachomati...
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