trachomatis infections were measured following treatment with four anti- Chlamydia molecules (tetracycline, polymyxin B, hydroxyethyl cellulose, and anti- C. For evaluating the efficacy and potential of iBAChIE to measure antichlamydial compounds, C. The accuracy and expediency of the automated method were compared to those of the conventional visual inspection in a 96-well format. To validate iBAChIE, the commonly utilized laboratory strain (LGV-2) and the more clinically relevant strain (serovar D) of C. In this study, image-based automated chlamydial identification and enumeration (iBAChIE) was developed using CellProfiler and CellProfiler Analyst. CellProfiler Analyst contains a supervised machine-learning system that is intuitive for development of image analysis algorithms via visual perception using a user-friendly interface ( 9, 10). The complement software, CellProfiler Analyst, performs analysis of image-derived quantitative information as defined within a very large collection of image-feature measurements (i.e., a cytological profile) produced by CellProfiler.
Cellprofiler analysing a set of images manual#
The limitations of manual enumeration are evident the process is labor intensive, field selection is prone to subjectivity, and results are based on a relatively restricted data set.įreely available open-source computational image analysis software, CellProfiler ( ), has been shown to be an effective tool for quantifying visual information in a variety of biological images, particularly in large-scale imaging experiments ( 2). The established procedure depends on visual inspection of the intracellular vacuole formed by Chlamydia, termed an inclusion, typically within a relatively low number of fields (e.g., 3 to 10). As a result, the direct immunofluorescent assay (IFA) is predominately relied on for accurate detection of Chlamydia infection. The paucity of high-throughput screening methods is heavily influenced by the obligate intracellular growth characteristics and the inability for axenic cultivation of Chlamydia.
trachomatis infections however, a vaccine or vaginal microbicide effective in preventing Chlamydia infection is currently unavailable for clinical use.Ī major limitation in the development of antichlamydial compounds is the lack of a well-established method for quantifying Chlamydia infection that is adaptable to a high-throughput screening format. Prevention strategies are essential given the high rate of asymptomatic C. Rogers, presented at the 9th International Symposium on Human Chlamydial Infection, International Chlamydia Symposium, San Francisco, CA, 1998). While current antibiotics are effective, resistance to the clinically preferred antibiotic (i.e., doxycycline) has already been observed in a Chlamydia species, albeit a nonhuman isolate ( C. trachomatis infection has been shown to associate with cervical cancer development and to contribute significantly to HIV transmission ( 8, 22). trachomatis infections in women are asymptomatic ( 19), and if left untreated, these infections can lead to serious complications, including pelvic inflammatory disease (PID), ectopic pregnancy, and infertility ( 5, 6, 15). Furthermore, iBAChIE is expected to be amenable to high-throughput screening studies for inhibitory compounds and fluorescently labeled molecules to study host-pathogen interactions.Ĭhlamydia trachomatis is the leading cause of bacterial sexually transmitted infection (STI) in the United States, for which novel preventative and therapeutic compounds are needed ( 3). Overall, these data support that iBAChIE is a highly effective tool for automated quantification of Chlamydia infection and assessment of antichlamydial activities of molecules. The inhibitory capability of established antichlamydial activity was also evaluated. The method yielded enumeration of different species and strains of Chlamydia highly comparably to the conventional manual methods while drastically reducing the analysis time.
Cellprofiler analysing a set of images software#
In this study, an immunofluorescence image-based method (termed image-based automated chlamydial identification and enumeration ) was customized for fully automated quantification of Chlamydia infection using the freely available open-source image analysis software program CellProfiler and the complementary data exploration software program CellProfiler Analyst. The immunofluorescence assay is the preferred approach for accurate quantification of the intracellular growth of Chlamydia. A major limitation in the identification of novel antichlamydial compounds is the paucity of effective methods for large-scale compound screening.
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