Aims: The ability to determine the presence and viability status of bacteria by molecular methods could offer significant advantages to the food, environmental and health sectors, in terms of improved speed and sensitivity of detection. Methods and Results: In this study, we have assessed three amplification techniques, PCR, RT‐PCR and NASBA, for their ability to detect nucleic acid persistence in an E. coli strain following heat‐killing. NASBA offered the greatest sensitivity of the three methods tested. The presence of residual DNA and mRNA could be detected by PCR and NASBA, respectively, for up to 30 h postdeath, by which time cell death had been confirmed by culture methods. Thus a single quantitative measurement based on nucleic acid amplification did not permit unequivocal determination of cell viability. Conclusions, Significance and Impact of the Study: The correlation between cell viability and persistence of nucleic acids must be well characterized for a particular analytical situation before molecular techniques can be substituted for traditional culture methods.

We have examined the in vivo and in vitro susceptibility of lymphocyte subpopulations to human T-lymphotropic virus type II (HTLV-II) to determine the cellular tropism for this virus. Monoclonal antibodies to T-cell subsets were used to separate highly purified CD4+ and CD8+ cells from peripheral blood lymphocytes of 35 individuals previously shown to be infected with HTLV-II. The purified T-cell subsets were analyzed for HTLV-II provirus (pol and tax gene sequences) by polymerase chain reaction (PCR) and cultured to determine virus expression by p24gag antigen detection. On the basis of PCR amplification in the pol and tax gene regions, both CD8+ subsets (89 to 91%) and CD4+ subsets (54 to 80%) from most infected subjects demonstrated HTLV-II provirus, irrespective of the viral genotype. Analysis of cultured lymphocytes demonstrated a higher spontaneous lymphocyte proliferation (17,986 +/- 4675 cpm) and p24gag antigen production (median 115 pg/ml; range 14-1360 pg/ml) in CD8+ cells compared to CD4+ cells (2333 +/- 826 cpm; p24gag antigen; 9 pg/ml; 2-250 pg/ml), suggesting a higher proviral load in CD8 cells. Limiting cell-dilution PCR analysis indicated that the CD8+ subset carried a higher HTLV-II provirus burden than the CD4+ subset. In vitro infection of purified CD4+ and CD8+ lymphocytes with irradiated HTLV-II cell lines resulted in productive infection of both subsets. Cell sorting and PCR analysis of lymphocyte subsets from 4 HTLV-II-infected subjects further demonstrated that in addition to CD4+ and CD8+ subsets, both CD45RO+ and CD45RO- and non-T-cells (CD14, CD16, and CD19) carried HTLV-II provirus. Taken together, these data suggest that HTLV-II possesses a broad tropism for peripheral blood mononuclear cells.

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We evaluated a cytofluorometric method for determining the number of antigens expressed on the cell surface of human lymphocytes. Using beads that have a known number of binding sites for mouse immunoglobulin and monoclonal antibodies specific for various antigens on human lymphocytes, we found that this system is quite reproducible, reliable and technically easy to perform. The greatest source of variation in expression of cell surface antigens is interdonor variability.