Performances of automated digital imaging of Gram-stained slides with on-screen reading against manual microscopy.

The objective of this study was to evaluate the performances of the automated digital imaging of Gram-stained slides against manual microscopy. Four hundred forty-three identified Gram-stained slides were included in this study. When both methods agreed, we considered the results as correct, and no further examination was carried out. Whenever the methods gave discrepant results, we reviewed the digital images and the glass slides by manual microscopy to avoid incorrectly read smears. The final result was a consensus of multiple independent reader interpretations. Among the 443 slides analyzed in this study, 101 (22.8%) showed discrepant results between the compared methods. The rates of discrepant results according to the specimen types were 5.7% (9/157) for positive blood cultures, 42% (60/142) for respiratory tract specimens, and 22% (32/144) for sterile site specimens. After a subsequent review of the discrepant slides, the final rate of discrepancies dropped to 7.0% (31/443). The overall agreement between the compared methods and the culture results reached 78% (345/443) and 79% (349/443) for manual microscopy and automated digital imaging, respectively. According to culture results, the specificity for automated digital imaging and manual microscopy were 90.8% and 87.7% respectively. In contrast, sensitivity was 84.1% for the two compared methods. The discrepant results were mostly encountered with microorganism morphologies of rare occurrence. The results reported in this study emphasize that on-screen reading is challenging, since the recognition of morphologies on-screen can appear different as compared to routine manual microscopy. Monitoring of Gram stain errors, which is facilitated by automated digital imaging, remains crucial for the quality control of reported Gram stain results.

Antagonism to human BST-2/tetherin by Sendai virus glycoproteins.

Tetherin is an interferon-inducible factor that restricts viral particle production. We show here that Sendai virus (SeV) induces a drastic decrease in tetherin levels in infected HeLa cells. Using ectopic expression of tetherin in Madin-Darby canine kidney cells, we find that infectious SeV production is sensitive to restriction by tetherin, suggesting that SeV downregulates tetherin to counter this form of cellular restriction. By using radioactive tetherin in pulse-chase experiments, applying conditions that limit protein degradation, and by estimating tetherin mRNA levels, we find that tetherin degradation is the mechanism of downregulation. Suppression of the virus envelope proteins matrix, fusion (F) or haemagglutinin-neuraminidase protein (HN) during the course of infection demonstrates that F and HN, in concert, are responsible for tetherin degradation. The mechanism(s) by which these two viral glycoproteins participate in degrading tetherin remains to be determined.