The aim of this study was to compare the effectiveness of two methods of antibiotic susceptibility testing performed on Chlamydia trachomatis-infected cells: a flow cytometric detection method and the standard method, which consists of a microscopic reading of minimal inhibitory concentration (MIC). The L2 reference strain and 13 clinical strains isolated from six patients presenting recurrent infections were tested. McCoy cells infected with an inoculum of 10(5) inclusion forming units (IFU)/ml were incubated with serial dilutions of doxycycline, ofloxacin, and erythromycin. Mean fluorescence intensity (MFI) of cells was determined by flow cytometry after staining of chlamydial inclusions with an anti-Chlamydia fluorescent monoclonal antibody. The end-point values determined by flow cytometry and microscopic reading were equivalent but presented the same imprecision. Calculation of the inhibitory concentration 50 (IC50) by flow cytometry, defined as the antibiotic concentration required to reduce the drug-free control MFI by 50%, allowed a more objective and precise evaluation of antibiotic activity than MIC. Moreover, IC50 values were reproducible, independent of the antibiotic dilution series tested, and could be used to compare the in vitro efficiency of various drugs on C. trachomatis. No resistant strain was found among the 13 clinical isolates of C. trachomatis tested.
We studied the feasibility and the sensitivity of fluorescent in situ hybridization (FISH) using leukemic or host/donor-specific probes on flow-sorted cells to assess minimal residual disease (MRD) or chimerism in transplanted patients in complete remission. We first performed experimental models of MRD and chimerism by mixing HL60 cells and normal lymphocytes in different proportions. Over 80% HL60 cells were obtained from mixtures of 5% HL60 cells in peripheral blood mononuclear cells (PBMC). We then evaluated MRD and mixed chimerism in a chronic myelogenous leukemia patient in relapse after allogeneic sex-mismatched bone marrow transplantation (BMT), who had received a donor lymphocyte infusion (DLI). Three months after DLI, mixed chimerism was observed in each bone marrow (BM)-sorted lineage (CD13+, CD14+, CD20+, and CD3+), with the highest level of recipient cells in the granulocytic lineage (CD13+). Five months after DLI, host cells were at a low level but remained detectable in the granulocytic lineage. In the same sample, the bcr-abl gene was detected in the granulocytic lineage and not in the lymphocytes. We also studied chimerism in an aplastic anemia sex-mismatched transplanted female patient. We determined the proportion of recipient total lymphocytes, CD4+ and CD8+ lymphocytes, and CD14+ monocytes under cyclosporin A therapy on five peripheral blood samples and one BM sample over 5 months. Results showed a regular decrease in recipient total lymphocytes (26.6% to 10.6%) and monocytes (20.7% to 8%). CD8(+)-recipient cells decreased rapidly, while CD4+ remained stable (17%). This work demonstrates the feasibility of FISH after cell sorting, combining the sensitivities of both flow cytometry and FISH and the specificities of both immunophenotyping and genotype analysis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.