Preterm premature rupture of membranes without labor is not associated with increased levels of matrix metalloproteinase-9 protein.

OBJECTIVES: Preterm premature rupture of membranes (PROM) accounts for 30-40% of all preterm births. The objectives of this study were to determine whether matrix metalloproteinase-9 (MMP-9) is increased in preterm PROM fetal membranes, whether labor or gestational age affects expression, and whether the increase is localized to the rupture site or is membrane-wide. METHODS: Fetal membranes were collected from 15 pregnancies complicated by preterm PROM and 26 control cases, which delivered at term or preterm without PROM. The preterm PROM cases represented both patients who labored and those who did not. Membrane samples at the rupture site and a remote site (approximately > 5 cm) were analyzed for MMP-9 protein and enzymatic activity by Western blot and gelatin zymography, respectively. RESULTS: MMP-9 levels in fetal membranes were similar at both the rupture and the remote sites. The highest levels of total MMP-9 protein were found in preterm PROM patients with labor (p < 0.05) and were increased four-fold over protein levels in non-laboring preterm PROM patients delivered by Cesarean section (p < 0.001). In preterm PROM patients without labor, levels of MMP-9 protein were similar to those of non-laboring patients at term and preterm. Zymography correlated with protein results in all membranes. CONCLUSIONS: Preterm PROM without labor is not associated with increased membrane levels of MMP-9 protein, suggesting that its local elevation does not play a role in early membrane rupture.

Levels of vaginal secretory leukocyte protease inhibitor are decreased in women with lower reproductive tract infections.

OBJECTIVE: Secretory leukocyte protease inhibitor contributes resistance to primary human immunodeficiency virus infection in the oral cavity. However, the levels of this inhibitor in the genital tract of women with sexually transmitted diseases or vaginitis are not well described. The objective was to determine vaginal inhibitor levels in women with symptomatic and asymptomatic genital infections. STUDY DESIGN: We tested 207 nonpregnant women for Neisseria gonorrhoeae, Trichomonas vaginalis, Chlamydia trachomatis, Candida species, and bacterial vaginosis by standard methods. A second group of symptom-free pregnant women (N = 231) was also studied. Secretory leukocyte protease inhibitor was measured by enzyme-linked immunosorbent assay, and results were compared by nonparametric methods. RESULTS: Vaginal levels of secretory leukocyte protease inhibitor in both groups were significantly lower in women with any sexually transmitted disease than in those without infection (P<.0001). Patients with bacterial vaginosis and those with bacterial vaginosis with yeast vaginitis also had decreased levels (P<.025). CONCLUSIONS: Levels of secretory leukocyte protease inhibitor in vaginal fluid are decreased in women with lower genital tract infection. This may represent a common mechanism of increasing susceptibility to infection with human immunodeficiency virus.

Evaluation of a deoxyribonucleic acid probe for the detection of Trichomonas vaginalis in vaginal secretions.

OBJECTIVE: Our purpose was to compare Affirm VP, a new deoxyribonucleic acid probe test, with standard "wet preparation" microscopic examinations and culture for the identification of Trichomonas vaginalis organisms in vaginal secretions. STUDY DESIGN: We examined vaginal samples from 615 women with symptoms or signs of vaginitis for T. vaginalis using the deoxyribonucleic acid probe test, microscopic examination of wet preparations of vaginal secretions, and culture in modified Diamond's medium. RESULTS: T. vaginalis was identified in specimens from 95 (15.4%) of the 615 patients. Cultures in Diamond's medium identified 93 (98%) of the 95 infected patients. Vaginal wet preparation identified 76 (80%) of the infected women. The deoxyribonucleic acid probe test detected 86 (90.5%) of the 95 infected patients. There was one false-positive deoxyribonucleic acid probe test (specificity 519/520: 99.8%). CONCLUSION: The Affirm VP deoxyribonucleic acid probe test had a sensitivity of 90% and a specificity of 99.8% for the identification of T. vaginalis organisms in women with symptoms with a high prevalence of trichomoniasis. Such a nonculture test may be of considerable benefit in diagnosing T. vaginalis infections, especially in settings where microscopy, culture, or both are unavailable, inconvenient, or unreliably performed.

Identification of Mycoplasma binding proteins utilizing a 100 kilodalton lung fibroblast receptor.

A 100 kilodalton glycoprotein receptor for Mycoplasma pneumoniae has been isolated from MRC-5 human lung fibroblasts. This receptor, as well as anti-receptor serum, were both capable of inhibiting the attachment of 14C-labelled M. pneumoniae to MRC-5 fibroblasts. The receptor was also capable of inhibiting the attachment of C-labelled M. gallisepticum and M. genitalium, but not M. pulmonis, to MRC-5 fibroblasts. This indicates that a common sequence may exist in these binding proteins of M. pneumoniae, M. genitalium, and M. gallisepticum. This receptor and anti-receptor serum were utilized to probe M. pneumoniae, M. genitalium, and M. gallisepticum for their corresponding binding proteins. A 32 kilodalton protein in M. pneumoniae, a 90 kilodalton protein in M. genitalium and a 139 kilodalton protein in M. gallisepticum were recognized.

Gonococcal attachment to eukaryotic cells.

The attachment of Neisseria gonorrhoeae to eukaryotic cells grown in tissue culture was analyzed by use of light and electron microscopy and by labeling of the bacteria with [3H]- and [14C]adenine. Isogenic piliated and nonpiliated N. gonorrhoeae from opaque and transparent colonies were studied. The results of light microscopy studies showed that the gonococci attached to cells of human origin, including Flow 2000, HeLa 229, and HEp 2. Studies using radiolabeled gonococci gave comparable results. Piliated N. gonorrhoeae usually attached in larger numbers than nonpiliated organisms, and those from opaque colonies attached more often than isogenic variants from transparent colonies. Day-to-day variation in rate of attachment was observed. Scanning electron microscopy studies showed the gonococcal attachment to be specific for microvilli of the host cells. It is concluded that more N. gonorrhoeae from opaque colonies, as compared with isogenic variants from transparent colonies, attach to eukaryotic cells grown in tissue culture.

Etiology of acute salpingitis: influence of episode number and duration of symptoms.

The role of Neisseria gonorrhoeae in the etiology and pathogenesis of acute salpingitis and its relationship to nongonococcal salpingitis were investigated. To accomplish this goal, isolated microorganisms obtained from the fallopian tubes and cul-de-sac via laparoscopy were evaluated in relation to the number of episodes of salpingitis, duration of symptoms, and phase of menstrual cycle at infection onset. The incidence of isolation of N gonorrhoeae was inversely proportional to the number of episodes of salpingitis. No isolation of the gonococcus occurred from patients with 3 or more previous episodes of salpingitis. N gonorrhoeae was the most frequent organism recovered within the initial 24 hours of symptoms. Beyond 48 hours, the most frequent isolates were anaerobic bacteria, especially anaerobic cocci. Anaerobic bacteria were also recovered from the fallopian tubes in patients having their initial episode of salpingitis and within 24 hours of onset of symptoms. All fallopian tube isolates of gonococci were recovered within 7 days of the onset of menses.

Auxotypes and antibiotic susceptibilities of Neisseria gonorrhoeae from women with acute salpingitis. Comparison with gonococci causing uncomplicated genital tract infections in women.

An investigation of the MICs of various antibiotics and the nutritional requirements (auxotypes) of Neisseria gonorrhoeae recovered from the cervix, fallopian tubes, and peritoneal cavity of women with acute salpingitis was done. These MICs and auxotypes were compared to those of gonococci isolated from the cervix of women with uncomplicated or asymptomatic genital tract gonorrhea. The MICs of minocycline, penicillin G, ampicillin, cefoxitin, and cefaclor for gonococci isolated from women with acute salpingitis were significantly higher. Significant differences in auxotype patterns were identified between isolates from salpingitis cases and uncomplicated cases. The prototrophic form was the most common (75%) among salpingitis strains. No strains requiring arginine, hypoxanthine, and uracil were identified among the salpingitis isolates. In contrast, strains that required these nutrients were the most frequent auxotype among isolated from women with uncomplicated genital tract gonorrhea. Discriminant analysis revealed that the MIC of minocycline and the auxotype were the most powerful discriminators between groups of patients with different manifestations of gonorrhea.

Microbiology and pathogenesis of acute salpingitis as determined by laparoscopy: what is the appropriate site to sample?

Acute salpingitis is a polymicrobial disease. Neisseria gonorrhoeae and anaerobic gram-positive cocci were the predominant microorganisms isolated from the fallopian tubes of salpingitis patients. Gonococci were isolated from the fallopian tubes in eight of 35 (23%) patients; anaerobic bacteria were recovered from 10 of 35 (28.5%). Although Chlamydia trachomatis was not recovered from the fallopian tube exudate, there was abundant serologic evidence of chlamydial infection in the salpingitis patients. Twenty-three percent of patients with paired sera had a fourfold rise in IgM and IgG titer, which was consistent with systemic chlamydial infection. Comparison of cultures obtained via laparoscopy and culdocentesis suggested that culdocentesis is not an accurate reflection of the microbial milieu in the fallopian tube.

Scanning electron microscopy of attachment of Neisseria gonorrhoeae colony phenotypes to surfaces of human genital epithelia.

Attachment of different gonococcal colony phenotypes to explants of human genital tract epithelium was studied by means of scanning electron microscopy and radioisotope-labeled gonococci. Heavily piliated organisms attached in greater numbers than nonpiliated organisms. Gonococci from transparent colony phenotypes attached in higher numbers than gonococci from opaque phenotypes to all tissues studied. Transitional cells from cervical tissues showing a gradual squamocolumnar transition demonstrated more gonococci attached per surface area than either endocervical or fallopian tube epithelium. Squamous epithelium showed the fewest number of attached gonococci. In all tissues, the attachment of the gonococcus was to the tips and surfaces of the microvilli. Gonococcal colony phenotypes as well as the length and location of the cervical transition zone may influence the progression of cervical gonococcal infection to invasive disease.

In vitro modeling of acute salpingitis caused by Neisseria gonorrhoeae.

Normal human fallopian tube organ culture was used as an in vitro model to study Neisseria gonorrhoeae infections. The effects of various gonococcal colony phenotypes on morphology of the epithelium were studied by scanning electron microscopy. After 30 minutes' incubation, there was striking attachment of piliated transparent phenotypes to the epithelium; however, there was no obvious pathology. After 24 hours' incubation, there were microcolony formation, slight swelling and hyperplasia of the mucosa, and occasional focal necrosis and sloughing of ciliated cells. Tissue from acute salpingitis showed widespread destruction of mucosa, hyperplasia, and crypt formation. Duplication of these findings in vitro may require longer incubation and the addition of other host factors.

Comparison of virulence markers of peritoneal and fallopian tube isolates with endocervical Neisseria gonorrhoeae isolates from women with acute salpingitis.

Neisseria gonorrhoeae strains which cause acute salpingitis are presumed to ascend the genital tract from the cervix. Previous studies utilized isolates obtained from endocervical canal cultures, although it was not known if the isolates truly represented the organisms present in the fallopian tubes. In this study, we compared N. gonorrhoeae isolates from endocervical canal cultures with fallopian tube or peritoneal cul-de-sac isolates or isolates from both sites obtained at laparoscopy. Potential virulence markers were studied, including colony phenotype, auxotype, antimicrobial agent susceptibility, protein patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and susceptibility to normal human serum. Six of seven cervical isolates had the same antibiograms and molecular weight for major outer membrane proteins as those of the corresponding peritoneal isolates. Auxotypes also were the same and included prototrophic, proline-requiring, and proline-and-arginine-requiring isolates. The isolates as a group appeared to be very susceptible to the bactericidal action of pooled serum from normal women. Colony phenotypes varied between sites; the fallopian tubecul-de-sac isolates were predominantly of transparent phenotype and piliated. The cervical isolates were either mixtures of equal quantities of opaque and transparent phenotypes or predominantly opaque phenotype. By these markers, patients' N. gonorrhoeae cervical isolates appeared to be the same as their isolates from fallopian tubes except for a difference or shift in colony phenotype.

Use of laparoscopy to determine the microbiologic etiology of acute salpingitis.

To determine the microbiologic etiology of acute salpingitis, laparoscopy was used in 26 patients to obtain specimens for a variety of microorganisms directly from the fallopian tube. Simultaneous culdocentesis was performed to obtain peritoneal fluid for microbiologic analysis. A variety of microorganisms were isolated from the fallopian tubes and cul-de-sac aspirate. However, the organisms isolated from the fallopian tube were not consistent with the cul-de-sac isolates. It appears that direct culture from the fallopian tube may be necessary to determine the microbiologic etiology and pathogenesis of acute salpingitis. N. gonorrhoeae was isolated from the cul-de-sac in 32 per cent of cases and the fallopian tube in 19 per cent. In patients with endocervical gonorrhea, the gonococcus was isolated from the fallopian tube in 38.5 per cent of cases. Aerobic and/or anaerobic bacteria were present in the cul-de-sac aspirate in 46 per cent of patients and in the fallopian tube in 38 per cent.

Aerobic and anaerobic susceptibility tests with three tetracyclines. Reassessment of the "class concept" of disk testing.

Both aerobic and anaerobic susceptibility tests were performed with tetracycline, doxycycline, and minocycline, using disk diffusion and agar dilution technics. The data were examined in order to reassess the concept of testing a single disk, representative of the tetracycline class of antimicrobics. All strains that were susceptible to a tetracycline disk were predictably susceptible to the tetracycline analogs. Some strains that gave zones in the resistant or intermediate range were susceptible or moderately susceptible to doxycycline and minocycline by agar dilution methods. However, disk tests with the more active analogs were often unproductive, since most tetracycline-resistant strains gave indeterminate results with doxycycline or minocycline disks. It was concluded that the "class concept" of disk testing is still appropriate and that tests with tetracycline disks predict susceptibility to the other tetracycline analogs reasonably well.

Rapid identification of Bacteroides fragilis with bile and antibiotic disks.

A simple screening test is described for separating Bacteroides fragilis from other anaerobic gram-negative bacilli. The test utilizes filter paper disks impregnated with 25 mg of oxgall (Difco), tested in conjunction with antibiotic identification disks. The bile disks and antibiotic disks are placed on a supplemented brucella blood agar plate which has been inoculated by swabbing with a standardized cell suspension. After 24 h at 35 degrees C in a GasPak jar, resistance to kanamycin and bile is taken as a presumptive identification of B. fragilis. Susceptibility to one or both disks indicates the need for further identification and additional biochemical tests are required. Those strains that produce insufficient growth within 24 h are not likely to be B. fragilis. The reliability of the bile disk method was tested by comparing results with 100 clinical isolates versus results with bile in thioglycolate broth, peptone-yeast extract-glucose broth, and tryptic soy agar. All four bile test methods gave equilvalent results, but the broth media required much longer periods of incubation.