Analysis of Shiga toxin subunit association by using hybrid A polypeptides and site-specific mutagenesis.

Shiga toxin (STX), a bacterial toxin produced by Shigella dysenteriae type 1, is a hexamer composed of five receptor-binding B subunits which encircle an alpha-helix at the carboxyl terminus of the enzymatic A polypeptide. Hybrid toxins constructed by fusing the A polypeptide sequences of STX and Shiga-like toxin type II were used to confirm that the carboxyl terminus of the A subunits governs association with the B pentamers. The alpha-helix of the 293-amino-acid STX A subunit contains nine residues (serine 279 to methionine 287) which penetrate the nonpolar pore of the B-subunit pentamer. Site-directed mutagenesis was used to establish the involvement of two residues bordering this alpha-helix, aspartic acid 278 and arginine 288, in coupling the C terminus of StxA to the B pentamer. Amino acid substitutions at StxB residues arginine 33 and tryptophan 34, which are on the membrane-contacting surface of the pentamer, reduced cytotoxicity without affecting holotoxin formation. Although these B-subunit mutations did not involve receptor-binding residues, they may have induced an electrostatic repulsion between the holotoxin and the mammalian cell membrane or disrupted cytoplasmic translocation.

Evaluation of two rapid tests for the diagnosis of Chlamydia trachomatis genital infections.

The performance of two new enzyme immunoassays (EIA) for the detection of Chlamydia trachomatis in a practice setting was compared. A consecutive series of 207 female patients seen at an inner-city sexually transmitted disease clinic were tested by cell culture, the Kodak SureCell (SC) and Abbott TestPack Chlamydia (TP) EIAs. In addition 210 male patients, selected by physicians on the basis of the fact that multiple urethral samples could be obtained, were tested by cell culture and SC. The prevalence of infection was 19% in the females and 12.5% in males. The sensitivity, specificity, positive predictive value and negative predictive value for the SC and TP were 88%, 95%, 81%, 97% and 59%, 99%, 95%, 91%, respectively, in the female population. The sensitivity of the SC was significantly greater than that of the TP (p less than or equal to 0.002). The performance values of the SC in men (in the same order) were 64%, 96%, 71% and 95%, respectively. The SC in male patients and the TP in female patients had low sensitivity. The sensitivity of the SC in female patients was significantly higher than that of the TP. However, the SC yielded more false positive results. To determine the utility of these tests in a practice setting further studies are required.

Comparison of the Kallested Pathfinder EIA, cytocentrifuged direct fluorescent antibody, and cell culture for the detection of Chlamydia trachomatis.

A total of 203 duplicate endocervical samples collected from patients at an adolescent health care center were tested for the presence of Chlamydia trachomatis by cell culture, Pathfinder enzyme immunoassay (EIA) (Kallestad) and cytocentrifuged direct fluorescent antibody (DFA). Compared to cell culture, the Pathfinder assay demonstrated a sensitivity and specificity of 85.2% and 100%, whereas the DFA procedure demonstrated to be 92.6% sensitive and 99.4% specific.

Comparison of three new nonculture tests in the diagnosis of Chlamydia genital infections.

This study compares three new rapid nonculture tests to cell culture with passage for the diagnosis of Chlamydia genital infections in sexually active adolescent and young adult females. Two hundred consecutive patients having a pelvic examination had cervical samples taken for the following: Papanicolaou smear, gonorrhea culture, Chlamydia cell culture, direct fluorescent antibody (DFA; Difco), isotopic DNA probe (Gen-Probe), and enzyme immunoassay (EIA; IDEIA III, Novo BioLabs). After resolution of discrepant results, 25 of the specimens were judged to be positive. The DFA identified 17 of the 25, with 3 false-positive results; the DNA probe identified 20 of the 25, with no false positive results; and the EIA identified 22 of the 25, with one false-positive result. The sensitivities, specificities, and positive and negative predictive values, respectively, were DFA, 68%, 98.2%, 85%, 95.5%; DNA probe, 80%, 100%, 100%, 97.2%; and EIA, 88%, 99.4%, 95.6%, 98.3%. These new rapid nonculture tests are comparable and relatively reliable, with trends favoring the EIA and the DNA probe. Further studies with larger samples are needed to determine their clinical utility.

Comparison of IDEIA III and cell culture for the detection of Chlamydia trachomatis in endocervical specimens.

A total of 803 endocervical samples were obtained from females with clinical or epidemiological histories suggesting chlamydia infection. These specimens were tested by IDEIA III and cell culture for the presence of Chlamydia trachomatis. After resolution of discrepant results by direct fluorescent-antibody staining of pelleted cell culture transport materials, IDEIA III demonstrated sensitivity, specificity, and positive and negative predictive values of 93.8, 99, 92.9, and 99.1%, respectively.

Comparison of DNA probe, monoclonal antibody enzyme immunoassay, and cell culture for the detection of Chlamydia trachomatis.

A total of 201 endocervical specimens were obtained from patients with a clinical or epidemiological history suggestive of chlamydial infection. These specimens were tested by DNA probe (Gen-Probe, San Diego, Calif.) and the IDEIA III (Boots-Celltech, Berkshire, United Kingdom) monoclonal antibody enzyme immunoassay and compared with cell culture for detection of Chlamydia trachomatis. Discrepancies between cell culture and antigen detection methods were resolved by direct fluorescent-antibody testing. In a population with a 17.4% prevalence, the sensitivities and specificities of these assays were 82.8 and 99.4%, respectively, for the DNA probe assay and 97.1 and 98.1%, respectively, for the IDEIA III.