Real-Time PCR-Based Mismatch Amplification Mutation Assay for Specific Detection of CS6-Expressing Allelic Variants of Enterotoxigenic Escherichia coli and Its Application in Assessing Diarrheal Cases and Asymptomatic Controls

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization factor CS6 is widespread in many developing countries, including India. The different allelic variants of CS6, caused by point mutations in its structural genes, cssA and cssB, are designated AIBI, AIIBII, AIIIBI, AIBII, and AIIIBII. A simple, reliable, and specific mismatch amplification mutation assay based on real-time quantitative PCR (MAMA-qPCR) was developed for the first time for the detection of CS6-expressing ETEC, along with the identification of allelic variations. The assay was based on mismatched nucleotide incorporation at the penultimate base at the 3' ends of the reverse primers specific for cssA and cssB and was validated using 38 CS6-expressing ETEC isolates. This strategy was effective in detecting all the alleles containing single-nucleotide polymorphisms. Using MAMA-qPCR, we also tested CS6 allelic variants in 145 ETEC isolates from children with acute diarrhea and asymptomatic infections, with the latter serving as controls. We observed that the AIBI and AIIIBI allelic variants were mostly associated with cases rather than controls, whereas the AIIBII variants were detected mostly in controls. In addition, the AIBI and AIIIBI alleles were frequently associated with ETEC harboring the heat-stable toxin gene (est) alone or with the heat-labile toxin gene (elt), whereas the AIIBII allele was predominant in ETEC isolates harboring the elt gene. This study may help in understanding the association of allelic variants in CS6-expressing ETEC with the clinical features of diarrhea, as well as in ETEC vaccine studies

Antigenic Change in Human Influenza A(H2N2) Viruses Detected by Using Human Plasma from Aged and Younger Adult Individuals

Human influenza A(H2N2) viruses emerged in 1957 and were replaced by A(H3N2) viruses in 1968. The antigenicity of human H2N2 viruses has been tested by using ferret antisera or mouse and human monoclonal antibodies. Here, we examined the antigenicity of human H2N2 viruses by using human plasma samples obtained from 50 aged individuals who were born between 1928 and 1933 and from 33 younger adult individuals who were born after 1962. The aged individuals possessed higher neutralization titers against H2N2 viruses isolated in 1957 and 1963 than those against H2N2 viruses isolated in 1968, whereas the younger adults who were born between 1962 and 1968 possessed higher neutralization titers against H2N2 viruses isolated in 1963 than those against other H2N2 viruses. Antigenic cartography revealed the antigenic changes that occurred in human H2N2 viruses during circulation in humans for 11 years, as detected by ferret antisera. These results show that even though aged individuals were likely exposed to more recent H2N2 viruses that are antigenically distinct from the earlier H2N2 viruses, they did not possess high neutralizing antibody titers to the more recent viruses, suggesting immunological imprinting of these individuals with the first H2N2 viruses they encountered and that this immunological imprinting lasts for over 50 years