Multiplex Bead Assay for the Serological Surveillance of Measles and Rubella.

There is increasing interest in evaluating antibody responses to multiple antigen targets in a single assay. Immunity to measles and rubella are often evaluated together because immunity is provided through combined vaccines and because routine immunization efforts and surveillance for measles and rubella pathogens are combined in many countries. The multiplex bead assay (MBA) also known as the multiplex immunoassay (MIA) described here combines the measurement of measles- and rubella-specific IgG antibodies in serum quantitatively according to international serum standards and has been successfully utilized in integrated serological surveillance.

shinyMBA: a novel R shiny application for quality control of the multiplex bead assay for serosurveillance studies.

The multiplex bead assay (MBA) based on Luminex xMAP technology can be used as a tool to measure seroprevalence as part of population immunity evaluations to multiple antigens in large-scale serosurveys. However, multiplexing several antigens presents challenges for quality control (QC) assessments of the data because multiple parameters must be evaluated for each antigen. MBA QC parameters include monitoring bead counts and median fluorescence intensity (MFI) for each antigen in plate wells, and performance of assay controls included on each plate. Analyzing these large datasets to identify plates failing QC standards presents challenges for many laboratories. We developed a novel R Shiny application, shinyMBA, to expedite the MBA QC processes and reduce the risk of user error. The app allows users to rapidly merge multi-plate assay outputs to evaluate bead count, MFI, and performance of assay controls using statistical process control charts for all antigen targets simultaneously. The utility of the shinyMBA application and its various outputs are demonstrated using data from 32 synthetic xPONENT files with 3 multiplex antigens and two population serosurveillance studies that evaluated 1200 and 3871 samples, respectively, for 20 multiplexed antigens. The shinyMBA open-source code is available for download and modification at https://github.com/CDCgov/shinyMBA . Incorporation of shinyMBA into Luminex serosurveillance workflows can vastly improve the speed and accuracy of QC processes.

Development of a Measles and Rubella Multiplex Bead Serological Assay for Assessing Population Immunity.

Serosurveys are important tools for estimating population immunity and providing immunization activity guidance. The measles and rubella multiplex bead assay (MBA) offers multiple advantages over standard serological assays and was validated by comparison with the enzyme-linked immunosorbent assay (ELISA) and the measles plaque reduction neutralization (PRN) assay. Results from a laboratory-produced purified measles virus whole-virus antigen MBA (MeV WVAL) correlated better with ELISA and PRN than results from the baculovirus-expressed measles nucleoprotein (N) MBA. Therefore, a commercially produced whole-virus antigen (MeV WVAC) was evaluated. Serum IgG antibody concentrations correlated significantly with a strong linear relationship between the MeV WVAC and MeV WVAL MBAs (R = 0.962 and R2 = 0.926). IgG concentrations from the MeV WVAC MBA showed strong correlation with PRN titers (R = 0.846), with a linear relationship comparable to values obtained with the MeV WVAL MBA and PRN assay (R2 = 0.716 and R2 = 0.768, respectively). Receiver operating characteristic (ROC) curve analysis of the MeV WVAC using PRN titer as the comparator resulted in a seroprotection cutoff of 153 mIU/ml, similar to the established correlate of protection of 120 mIU/ml, with a sensitivity of 98% and a specificity of 83%. IgG concentrations correlated strongly between the rubella WVA MBA and ELISA (R = 0.959 and R2 = 0.919). ROC analysis of the rubella MBA using ELISA as the comparator yielded a cutoff of 9.36 IU/ml, similar to the accepted cutoff of 10 IU/ml for seroprotection, with a sensitivity of 99% and a specificity of 100%. These results support use of the MBA for multiantigen serosurveys assessing measles and rubella population immunity.

Life in the cystic fibrosis upper respiratory tract influences competitive ability of the opportunistic pathogen Pseudomonas aeruginosa.

Understanding characteristic differences between host-associated and free-living opportunistic pathogens can provide insight into the fundamental requirements for success after dispersal to the host environment, and more generally into the ecological and evolutionary processes by which populations respond to simultaneous selection on complex interacting traits. We examined how cystic fibrosis (CF)-associated and environmental isolates of the opportunistic pathogen Pseudomonas aeruginosa differ in the production of an ecologically important class of proteinaceous toxins known as bacteriocins, and how overall competitive ability depends on the production of and resistance to these bacteriocins. We determined bacteriocin gene content in a diverse collection of environmental and CF isolates and measured bacteriocin-mediated inhibition, resistance and the outcome of competition in a shared environment between all possible pairs of these isolates at 25°C and 37°C. Although CF isolates encoded significantly more bacteriocin genes, our phenotypic assays suggest that they have diminished bacteriocin-mediated killing and resistance capabilities relative to environmental isolates, regardless of incubation temperature. Notably, however, although bacteriocin killing and resistance profiles significantly predicted head-to-head competitive outcomes, CF and environmental isolates did not differ significantly in their competitive ability. This suggests that the contribution of bacteriocins to competitive ability involves selection on other traits that may be pleiotropically linked to interference competition mediated by bacteriocins.