Development of a Sensitive Chemiluminescent Neuraminidase Assay for the Determination of Influenza Virus Susceptibility to Zanamivir

Buxton, Rachel C.; Edwards, Brooks S.; Juo, Rouh R.; Voyta, John C.; Tisdale, Margaret; Bethell, Richard C.

doi:10.1006/abio.2000.4517

Cited by 119 publications

(75 citation statements)

References 19 publications

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“…The fluorescent NI assay (Table 3) generated higher IC 50 s than the chemiluminescent assay (Table 2), which was in accord with previous observations (38) and may be attributed to structural differences in the synthetic substrates utilized (6,32). In our experience and others' (3,29), the fluorescent NI assay offers a better discrimination between the IC 50 s of the mutant and wild-type viruses; however, it also requires a higher virus titer than the chemiluminescent assay and therefore necessitates additional propagation of viruses in MDCK cells.…”

Section: Vol 54 2010 Emergence Of Drug-resistant Influenza a Variansupporting

confidence: 89%

Detection of E119V and E119I Mutations in Influenza A (H3N2) Viruses Isolated from an Immunocompromised Patient: Challenges in Diagnosis of Oseltamivir Resistance

Okomo-Adhiambo

Demmler-Harrison

Deyde

et al. 2010

Antimicrob Agents Chemother

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The clinical use of the neuraminidase inhibitor (NAI) oseltamivir is associated with the emergence of drug resistance resulting from subtype-specific neuraminidase (NA) mutations. The influenza A/Texas/12/2007 (H3N2) virus isolated from an oseltamivir-treated immunocompromised patient exhibited reduced susceptibility to oseltamivir in the chemiluminescent neuraminidase inhibition (NI) assay (ϳ60-fold increase in its 50% inhibitory concentration [IC 50 ] compared to that for a control virus). When further propagated in cell culture, the isolate maintained reduced susceptibility to oseltamivir in both chemiluminescent and fluorescent NI assays (ϳ50-and 350-fold increases in IC 50 , respectively). Sequencing analysis of the isolate revealed a mix of nucleotides coding for amino acids at position 119 of the NA [E119(V/I)]. Plaque purification of the isolate yielded E119V and E119I variants, both exhibiting reduced susceptibility to oseltamivir. The E119I variant also showed decreased susceptibility to zanamivir and the investigational NAIs peramivir and A-315675. The emergence of E119V variants in oseltamivir-treated patients has been previously reported; however, the E119I mutation detected here is a novel one which reduces susceptibility to several NAIs. Both mutations were not detected in unpropagated original clinical specimens using either conventional sequencing or pyrosequencing, suggesting that these variants were present in very low proportions (<10%) in clinical specimens and gained dominance after virus propagation in MDCK cells. All virus isolates recovered from the patient were resistant to adamantanes. Our findings highlight the potential for emergence and persistence of multidrug-resistant influenza viruses in oseltamivir-treated immunocompromised subjects and also highlight challenges for drug resistance diagnosis due to the genetic instability of the virus population upon propagation in cell culture.

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Section: Vol 54 2010 Emergence Of Drug-resistant Influenza a Variansupporting

confidence: 89%

Detection of E119V and E119I Mutations in Influenza A (H3N2) Viruses Isolated from an Immunocompromised Patient: Challenges in Diagnosis of Oseltamivir Resistance

Okomo-Adhiambo

Demmler-Harrison

Deyde

et al. 2010

Antimicrob Agents Chemother

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“…In this prototype test, 0.9 ml of the respiratory specimen was added to a prefilter tube containing salts and detergent, which created an optimal milieu for influenza virus neuraminidase activity (2,4,18). Clarification of the specimen occurred at the prefilter tip as the specimen was added to the bottom half of a proprietary two-chamber reaction vessel containing an influenza virus neuraminidase-specific substrate and two light enhancers.…”

Section: Methodsmentioning

confidence: 99%

“…In chemistry analyzers, chemiluminescence is generally accepted as being more sensitive than enzyme immunoassays (14). Chemiluminescence provides an opportunity for increasing the sensitivity of rapid tests (1,4).…”

mentioning

confidence: 99%

Clinical Evaluation of the ZstatFlu-II Test: a Chemiluminescent Rapid Diagnostic Test for Influenza Virus

et al. 2002

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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...

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“…Generating knowledge on possible resistance in a timely manner may, therefore, benefit patient management. Phenotypic antiviral assays, such as MuNANA and NA-star, determine the susceptibility of influenza viruses to neuraminidase inhibitors by testing the activity of the viral enzyme neuraminidase under increasing inhibitor concentrations [2]. When knowledge on genotypic changes related to phenotypic resistance are lacking due to the introduction of a new neuraminidase inhibitor or influenza subtype, these assays generate valuable information needed for patient management.…”

Section: Satisfying the Need For Rapid Diagnosis Of New Variant Influmentioning

confidence: 99%

Satisfying the need for rapid diagnosis of new variant influenza A H1N1

Vries

Schutten

2010

Expert Review of Molecular Diagnostics

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Development of a Sensitive Chemiluminescent Neuraminidase Assay for the Determination of Influenza Virus Susceptibility to Zanamivir

Cited by 119 publications

References 19 publications

Detection of E119V and E119I Mutations in Influenza A (H3N2) Viruses Isolated from an Immunocompromised Patient: Challenges in Diagnosis of Oseltamivir Resistance

Detection of E119V and E119I Mutations in Influenza A (H3N2) Viruses Isolated from an Immunocompromised Patient: Challenges in Diagnosis of Oseltamivir Resistance

Clinical Evaluation of the ZstatFlu-II Test: a Chemiluminescent Rapid Diagnostic Test for Influenza Virus

Satisfying the need for rapid diagnosis of new variant influenza A H1N1

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