Monitoring influenza virus susceptibility to oseltamivir using a new rapid assay, iART.

A new rapid assay for detecting oseltamivir resistance in influenza virus, iART, was used to test 149 clinical specimens. Results were obtained for 132, with iART indicating 41 as 'resistant'. For these, sequence analysis found known and suspected markers of oseltamivir resistance, while no such markers were detected for the remaining 91 samples. Viruses isolated from the 41 specimens showed reduced or highly reduced inhibition by neuraminidase inhibition assay. iART may facilitate broader antiviral resistance testing.

Simulated Respiratory Secretion for Use in the Development of Influenza Diagnostic Assays.

Many assays have been developed for the detection of influenza virus which is an important respiratory pathogen. Development of these assays commonly involves the use of human clinical samples for validation of their performance. However, clinical samples can be difficult to obtain, deteriorate over time, and be inconsistent in composition. The goal of this study was to develop a simulated respiratory secretion (SRS) that could act as a surrogate for clinical samples. To this end, we determined the effects major respiratory secretion components (Na+, K+, Ca2+, cells, albumin IgG, IgM, and mucin) have on the performance of influenza assays including both nucleic acid amplification and rapid antigen assays. Minimal effects on the molecular assays were observed for all of the components tested, except for serum derived human IgG, which suppressed the signal of the rapid antigen assays. Using dot blots we were able to show anti-influenza nucleoprotein IgG antibodies are common in human respiratory samples. We composed a SRS that contained mid-point levels of human respiratory sample components and studied its effect compared to phosphate buffered saline and virus negative clinical sample matrix on the Veritor, Sofia, CDC RT-PCR, Simplexa, cobas Liat, and Alere i influenza assays. Our results demonstrated that a SRS can interact with a variety of test methods in a similar manner to clinical samples with a similar impact on test performance.

Analytical reactivity of 13 commercially available rapid influenza diagnostic tests with H3N2v and recently circulating influenza viruses.

OBJECTIVES: Rapid influenza diagnostic tests (RIDTs) used widely in clinical practice are simple to use and provide results within 15 minutes; however, reported performance is variable, which causes concern when novel or variant viruses emerge. This study's goal was to assess the analytical reactivity of 13 RIDTs with recently circulating seasonal and H3N2v influenza viruses, using three different viral measures. DESIGN: Virus stocks were characterized by infectious dose (ID50 ) and nucleoprotein (NP) concentration, diluted at half-log dilutions, and tested with each RIDT and real-time RT-PCR. RESULTS: Strong correlation was observed between NP concentration and RIDT reactivity; however, only weak correlation was seen with ID50 or Ct values. Only four RIDTs detected viral NP at the lowest dilution for all influenza A viruses (IAV). Influenza A viruses not detected by more than one RIDT had lower NP levels. Of the 13 RIDTs, 9 had no significant differences in reactivity across IAV when compared to NP levels. CONCLUSIONS: Previous reports of RIDT performance typically compare reactivity based on ID50 titers, which in this study correlated only weakly with proportional amounts of viral NP in prepared virus samples. In the context of the strong correlation of RIDT reactivity with NP concentration, H3N2v was found to be as reactive as seasonal circulating IAV. While these findings may not reflect clinical performance of these RIDTs, measuring NP concentration can be useful in the future to assess comparable reactivity of available RIDTs, or to assess reactivity with newly evolving or emerging viruses.

Characterization of two classes of pancreatic shock factors: functional differences exhibited by hydrophilic and hydrophobic shock factors.

Pancreatic tissue homogenate induces a powerful pathophysiologic response sufficient to produce lethal shock in a rat. However, limited progress has been made in the biochemical characterization of these pancreas-derived active factors or their mechanisms of action. It has been shown that the pancreas is a major source of these shock-inducing factors and that they are generated by pancreatic proteinases. Porcine pancreas was homogenized and the filtered homogenate was subjected to organic extraction both before and after incubation for 2.5 h at 37 degrees C. The aqueous and lipid extracts of pancreatic homogenates were collected and analyzed for their ability to activate human neutrophils and to induce lethal shock in the rat. Neutrophil activation, a presumed hallmark of shock, was determined by chemiluminescence and myeloperoxidase (MPO) release. Only the intact homogenate and lipid extracts stimulated the neutrophils, and the aqueous extracts proved to be inactive. Neutrophils exhibited enhanced cellular activation when exposed to substimulatory levels of either formyl-methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF) followed by substimulatory levels of the lipid extracts, but not by the aqueous extracts. Both the lipid and aqueous extracts induced dramatic decreases in heart rate and blood pressure when injected in the rat, often resulting in lethal shock. In all cases, incubation of the homogenates at 37 degrees C enhanced the potency of the extracts. Our results demonstrated that the pancreas-derived homogenate and lipid factors were capable of inducing both neutrophil activation and shock. These results support the hypothesis that shock is produced via neutrophils that have been activated by inflammatory components. However, the shock-inducing factors in the aqueous extracts (i.e., hydrophilic fraction of the homogenate) apparently function via a pathway independent of neutrophil activation. This is the first evidence that there are both hydrophobic and hydrophilic factors generated in tissue homogenates capable of inducing shock, and that these different chemical classes of factors appear to function via separate mechanisms.