[Urogenital chlamydial infections in women and men]. / Urogenitale Chlamydieninfektionen bei Frau und Mann.

Genital infections with Chlamydia trachomatis occur in all social groups in Germany. About 100,000 German women are sterile because of tubal scarring due to chlamydiae. Genital chlamydial infections are asymptomatic in 70% of patients, even if salpingitis occurs. Typical symptoms of chlamydial infection are purulent cervicitis with vaginal discharge, painful cervical bleeding because of endometritis, lower abdominal pain with dyspareunia, and upper abdominal pain because of perihepatitis. DNA amplification tests on first voided urine or cervical swab are the most sensitive routine tests. Specific serum antibodies to C. trachomatis indicate a previous infection in sterile women. For treatment, a 10-14 day course of doxycycline 200 mg daily or a macrolide antibiotic in the patient as well as in the sexual partner is recommended. In the male, C. trachomatis causes urethritis and epididymitis. Opinions differ about involvement of the prostate gland and seminal vesicles. Identification of C. trachomatis antigen or DNA in the accessory gland secretions is not sufficiently reproducible. The two vectors are easily diagnosed in urethral swabs or in urine. The occurrence of chlamydial antibodies in serum or in seminal fluid is not a sign of current infection. Reliable studies which indicate a reduced fertility of men infected with C. trachomatis are not available.

[Chlamydia and other sexually transmitted bacterial infections]. / Chlamydien und andere sexuell übertragene bakterielle Infektionen.

Chlamydia trachomatis is the most common sexually transmitted bacterium worldwide. In Western Europe, the prevalence of gonorrhoea has decreased by more than 95% since the 1970ies; "tripper" and syphilis are essentially confined to high-risk groups while genital chlamydial infections affect people of all social classes, but information about chlamydia is still scarce in many European countries. Clinically genital chlamydial infections resemble gonorrhoea (dysuria, discharge, irregular bleeding, dyspareunia, perihepatitis) and may be mistaken for appendicitis. However, Chlamydia trachomatis persists longer and more often asymptomatic than Neisseria gonorrhoeae in the urogenital tract of men and women. About 20% of all chlamydia infected women suffer from partial or complete tubal occlusion. Chlamydia trachomatis is the leading cause of female infertility, but most of these women never experienced any clinical sign of pelvic inflammatory disease. Since particle concentrations are often very low in urine and cervical secretions only DNA-amplification tests, e.g. PCR or LCR, exhibit sufficient sensitivity for direct detection Chlamydia trachomatis. While Neisseria gonorrhoeae is eradicated by single-shot treatment with commonly used antibiotics like penicillins or cephalosporins Chlamydia trachomatis affords treatment for at least 10 days with doxycyline or macrolides. Partner treatment is essential to avoid reinfections. Condoms not only protect against HIV, but also against chlamydia, gonorrhoea and syphilis.

Discordant prevalence of chlamydia trachomatis in asymptomatic couples screened using urine ligase chain reaction.

The following study was conducted to determine whether there would be an effect on the prevalence of Chlamydia trachomatis if both partners in a sexual relationship, rather than only one, underwent screening. First-void urine samples were collected from 1,690 asymptomatic women (mean age, 30 years; range, 15-70 years) and their male sex partners (mean age, 33 years; range, 16-71 years). The duration of sexual partnership for these subjects ranged from 2 months to more than 10 years.. At the time of testing, 687 of the women were pregnant. Ligase chain reaction testing revealed that 42 (2.5%) female and 63 (3.7%) male urine samples were positive. Detection rates for Chlamydia trachomatis differed for males and females, a difference that was found to be significant (P<0.0046, McNemar chi-square). Both partners tested positive in 27 (1.6%) couples, whereas at least one partner tested positive in 78 (4.6%) couples. Thus, screening males for Chlamydia trachomatis would have identified 63 (81%) of these 78 couples compared with only 42 (54%) couples had females been screened exclusively. In standard clinical practice, women most often undergo screening. The results of this study underscore the need to screen both males and females for Chlamydia trachomatis.

Chlamydia pneumoniae serology: interlaboratory variation in microimmunofluorescence assay results.

The lack of standardization in chlamydia serology has made interpretation of published data difficult. This study was initiated to determine the extent of interlaboratory variation of microimmunofluorescence (MIF) test results for the serodiagnosis of Chlamydia pneumoniae infections. Identical panels of 22 sera were sent to 14 laboratories in eight countries for the determination of IgG and IgM antibodies by MIF. Although there was extensive variation in the numeric titer values, the overall percentage agreement with the reference standard titers from the University of Washington was 80%. For results by serodiagnostic category, the best agreement was for four-fold rise in IgG titers, while the lowest agreement was for negative or low IgG titers. Agreement for IgM titers was 50%-95%. Four laboratories failed to discern false-positive IgM titers possibly because of the presence of rheumatoid factor. Further studies are underway to determine the source of interlaboratory variation for the MIF test.

Elevated gene expression of interleukin-8 in cord blood is a sensitive marker for neonatal infection.

Bacterial infection is a major cause of neonatal morbidity and mortality. Early diagnosis is essential for a successful treatment and outcome. Cytokine plasma levels are suggested to be sensitive parameters for the diagnosis of neonatal sepsis. The aim of this study was to assess cytokine mRNA expression in cord blood cells as a marker for neonatal infection. In a prospective study, cord blood samples of neonates with septic bacterial infection were analyzed qualitatively and semiquantitatively by reverse transcriptase-polymerase chain reaction (RT-PCR) for mRNA expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, as well as for IL-8 cord plasma levels. Results were compared to those of non-septic neonates. A method was used requiring only a microvolume (25 microl or less) of cord blood. Cord plasma levels of IL-8 were significantly elevated in septic infants (n = 9) when compared to infants with not confirmed sepsis (n = 22) and healthy infants that served as controls (n = 68) (median 1,686 vs 262.7 vs 33.1 pg/ml, P < 0.001). The presence of IL-6 and TNF-alpha gene expression was observed more frequently in septic than in non-septic patients; sensitivity, however, reached only 56 and 67%, respectively. When using a semiquantitative approach for analyzing IL-8 mRNA levels, a high sensitivity (86%) and specificity (96%) for the detection of sepsis was achieved. A new method for the early diagnosis of neonatal infection is described measuring cytokine mRNA in neonatal cord blood cells. With this molecular approach only a microvolume of blood is required for analysis.

Detection of seroconversion and persistence of Chlamydia trachomatis antibodies in five different serological tests.

Microimmunofluorescence (MIF), a Chlamydia trachomatis species-specific enzyme immunoassay incorporating lipopolysaccharide-extracted Chlamydia trachomatis L2 elementary bodies, two different synthetic peptide-based species-specific tests, and a recombinant lipopolysaccharide genus-specific test were performed on multiple follow-up sera (n = 104 total) from 16 women with Chlamydia trachomatis-positive cervical swabs. These women included five with IgG seroconversions, five with Chlamydia trachomatis reinfections after initial therapy, and six with serologic follow-up of more than 6 years after antibiotic therapy. Of all the tests employed in this study, MIF IgG reverted earliest to negative titers, while MIF IgA was the least sensitive. The lipopolysaccharide-extracted elementary body enzyme immunoassay exhibited the closest correlation with the MIF test. The highest test sensitivity was observed in one of the synthetic peptide-based tests, which detected earliest seroconversions and longest IgG persistence. The other synthetic peptide-based test gave false-negative results in 2 of 16 women and did not detect seroconversion earlier than the MIF test. Seroconversion and persistence of genus-specific IgG--cross-reactivity with Chlamydia pneumoniae--against lipopolysaccharide were similar to species-specific IgG. A significant serologic response to reinfection was observed only in women with signs of pelvic inflammatory disease. Species-specific tests of high sensitivity and reproducibility are best suited for gynecological diagnostic purposes.

Plasma levels and gene expression of granulocyte colony-stimulating factor, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-8, and soluble intercellular adhesion molecule-1 in neonatal early onset sepsis.

Bacterial sepsis is still a leading cause of neonatal morbidity and mortality. Early onset sepsis in particular, presents with a different clinical course and involves other pathogens than sepsis later in life. In this study, plasma concentrations and mRNA expression of granulocyte colony-stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6, IL-8, and soluble intercellular adhesion molecule-1 (sICAM-1) of neonates with early onset sepsis were evaluated in cord blood and during the first days of life. Irrespective of prematurity, plasma levels of G-CSF, TNF-alpha, IL-1beta, IL-6, and IL-8, but not sICAM-1, were excessively elevated in septic neonates when compared with both healthy infants and infants with clinically suspected but not confirmed sepsis. Compared with the corresponding maternal levels, neonatal cytokine cord plasma levels were likewise highly elevated, indicating the endogenous cytokine production by the neonate. With the exception of TNF-alpha, mRNA expression in blood cells from septic infants was, however, not more frequently detectable than in those from nonseptic patients. Cytokine levels decreased significantly within the first days of life, whereas levels of sICAM-1 and C-reactive protein increased during the same time period. In summary, in contrast to C-reactive protein and sICAM-1, cord blood plasma levels, but not the presence of mRNA, of G-CSF, TNF-alpha, IL-1beta, IL-6, and IL-8 can predict neonatal early onset sepsis with a high sensitivity and specificity. Cell types other than blood cells are likely to contribute considerably to the high cytokine production in septic newborns.

Seminal tract infections: impact on male fertility and treatment options.

Bacterial and viral infections of the genital tract may be important aetiological factors for male infertility. Infectious processes may lead to deterioration of spermatogenesis, impairment of sperm function and/or obstruction of the seminal tract. Detection of bacteria in semen does not necessarily signify infection since bacteriospermia may represent contamination, colonization or infection. Reported prevalence of Ureaplasma urealyticum in human semen varies from 10 to 40%. Enterobacteria can even be found in up to 90% of semen samples depending on the sensitivity of detection methods used. Chlamydia trachomatis is the most frequent sexually transmitted bacterial organism in industrialized countries. It is suggested that its main influence is due to sexual transmission resulting in tubal disease and subsequent infertility in the female partner rather than a direct influence on male reproductive functions. The effect of leukocytospermia on male fertility is controversial. This is probably due to different detection methods, different populations studied and to the fact that leukocyte subtypes in semen may have different functions. In addition to potentially negative effects, leukocytes may even have protective effects on spermatozoa. Only recently have amplification methods been established to detect viruses in semen with high sensitivity and specificity. It is unclear if these infections significantly contribute to male infertility.

Evidence of labile inhibitors in the detection of Chlamydia trachomatis in cervical specimens by polymerase chain reaction.

A total of 276 cervical swabs (241 from first visits and 35 from follow-up visits) from 241 women were tested for Chlamydia trachomatis by polymerase chain reaction (PCR) and enzyme immunoassay (EIA). Sixty-one smears (53 from first visits and 8 from follow-up visits) from 53 women were stained by direct fluorescent antibody (DFA). Twenty-one (8.7%) women had positive swabs in at least two different tests. All follow-up swabs (collected between 3 days and 3 weeks after the first clinical visit) were positive in at least one test when the woman had been positive at the first visit and no antibiotic treatment had been initiated. Including swabs from follow-up visits and DFA results, the respective sensitivities and specificities of the assays were as follows: PCR, 75.9% and 100%; EIA, 69% and 98.4%. The seven swabs that were false negative by PCR (tested initially after thawing from -20 degrees C) were mailed nonrefrigerated to the assay manufacturer, where they tested true positive. These data point to labile inhibitors of the PCR, predominantly cervical mucus.

Immune response to Chlamydia trachomatis heat-shock protein in infertile female patients and influence of Chlamydia pneumoniae antibodies.

A total of 446 sera from 245 patients with primary or secondary infertility, all of whom were examined laparoscopically, 117 patients with Chlamydia trachomatis-positive cervical swabs, and 84 control persons (50 obstetric patients and 34 female blood donors) were tested for antibodies to Chlamydia trachomatis and to Chlamydia pneumoniae with the microimmunofluorescence (MIF) test. MIF test antibody rates were highest in patients with complete tubal occlusion (73%) and in patients with proven Chlamydia trachomatis infection (74%), whereas only 9 to 10% of the control group showed Chlamydia trachomatis antibodies. Reaction to the 60 kDa antigen of Chlamydia trachomatis, a heat-shock protein (hsp) analogue, has been suggested as a possible marker for the development of chronic sequelae after Chlamydia trachomatis infection. Immunoblot analysis of 222 sera (169 infertility patients, 20 antigen-positive patients, and 33 mothers) showed a significantly higher anti-hsp antibody rate in patients with complete tubal occlusion than in infertility patients with normal fallopian tubes (76% vs. 19%, p < 0.001). The presence of antibodies not only to Chlamydia trachomatis but also to Chlamydia pneumoniae in the MIF test was associated with a significantly higher rate of anti-hsp antibodies and with complete tubal occlusion. This association did not appear to be due to cross-reactivity between Chlamydia pneumoniae and Chlamydia trachomatis antibodies in the MIF test.

Chlamydia trachomatis species specific serology: ImmunoComb Chlamydia bivalent versus microimmunofluorescence (MIF).

The ImmunoComb Chlamydia Bivalent IgG/IgA (Orgenics, Israel) is a new quantitative serologic test that employs LPS extracted Chlamydia trachomatis L2 and LPS extracted Chlamydia pneumoniae elementary bodies on two separate antigenic spots. The Bivalent C. trachomatis specific test results were compared with microimmunofluorescence (MIF), the gold standard of chlamydial species specific serology. For C. trachomatis IgG the Bivalent was highly concordant with the MIF: the rate of positive titres (IgG > or = 1:8) was 10% vs. 11% in 100 blood donors, 18% vs. 16% in 111 obstetric patients (6% antigen prevalence), 26% vs. 22% in sterile women with open (n = 54) and 86% vs. 84% with occluded (n = 51) tubes, and 88% vs. 85% in 103 women with C. trachomatis positive cervical smears. Surprisingly, the Bivalent differed considerably from the MIF in IgA prevalence: in obstetric patients (8% vs. 4%), sterile women with open (13% vs. 6%) and occluded (71% vs. 20%) tubes, and women with positive cervical smears (78% vs. 24%). Bivalent IgA appeared to be more sensitive than MIF IgA and showed a stronger correlation with positive cervical smears in obstetric patients (sensitivity 67% vs. 0%, specificity 95% vs. 96%, positive prediction 44% vs. 0%, negative prediction 98% vs. 94%) and with tubal occlusion in sterile women (sensitivity 71% vs. 20%, specificity 87% vs. 94%, positive prediction 84% vs. 77%, negative prediction 76% vs. 55%). MIF IgM was of little diagnostic help. Supplemental to the often difficult C. trachomatis antigen detection, the easily performed Bivalent IgG/IgA appears to be of great value in routine diagnosis of genital chlamydial infections.

Chlamydial serology in genital infections: ImmunoComb versus Ipazyme.

The ImmunoComb Chlamydia trachomatis IgG/IgA (Orgenics, Israel) is a new serologic test using C. trachomatis L2 elementary bodies (Washington Research Foundation, Seattle) as antigen. The Ipazyme IgG/IgA test (Savyon, Israel) employs whole cells with C. trachomatis L2 inclusions, i.e. elementary and reticulate bodies. Theoretically, the ImmunoComb is expected to be less cross-reactive (LPS) with Chlamydia pneumoniae than the Ipazyme (LPS and reticulate body group specific antigens). Compared with the Ipazyme, the ImmunoComb IgA showed both a higher positive predictive value (36% versus 25%) and sensitivity (67% versus 33%) for antigen detection in a control group of 100 post partum women with a 6% prevalence of C. trachomatis positive cervical smears. In sterility patients (45 cases with occluded and 53 with open fallopian tubes) the tube status was predicted by the ImmunoComb (Ipazyme) with 74% (72%) positive predictive value, 87% (80%) sensitivity, and 87% (81%) negative predictive value. IgG/IgA prevalence in 118 patients with C. trachomatis positive cervical smears was 85%/55% for the ImmunoComb and 84%/49% for the Ipazyme. The ImmunoComb is considerably faster and easier in handling and less subjective in reading than the Ipazyme.

Quantitative electroelution of oligonucleotides and large DNA fragments from gels and purification by electrodialysis.

We have designed a new device [Biotrap (Elutrap in the U.S.A. and Canada), available from Schleicher & Schuell] for electroelution, -concentration , and -dialysis of DNA and other charged macromolecules above 5 kDa. In an electric field, the DNA migrates in an open channel out of the gel slice through a microporous membrane, BT2, into the trap section, where it is retained by a very dense, non-adsorbant, and inert membrane BT1. Specifically designed for use in an electric field, the matrix of this new membrane is much denser than that of dialysis membranes. In contrast to dialysis membranes, BT1 will not adsorb large DNA fragments nor allow passage of small DNA fragments when subjected to an electric field. In the absence of an electric field, BT1 and BT2 effectively seal the trap, maintaining the final elution volume of the purified sample. The trap can contain from 200-600 microliter and is collected from above with a pipet. In the experiments described here, 85-95% of oligonucleotides (14-mer) and large (150 kb) DNA fragments were recovered, independent of fragment length. The purity of the eluted DNA was demonstrated by restriction enzyme digestion, nick-translation, primer extension, end-labeling with polynucleotide kinase, and ligation. Electrodialysis was successfully used for the complete removal of common contaminants inhibiting the polynucleotide kinase reaction and for the removal of CsCl from DNA samples.

Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap.

The membrane trap is a new device for the electroelution of all kinds of charged macromolecules from gels. Instead of dialysis membranes, the membrane trap uses a new membrane. Retention of macromolecules in an electric field by dialysis membranes depends on the presence of sodium dodecyl sulfate (SDS) in the buffer. The new membrane retains all charged macromolecules larger than approximately 5000 Da without adsorbing them, independent of the use of SDS. Here we report the electroelution of five different lipophilic membrane proteins (33 to 193 kDa) of Mycoplasma pneumoniae from preparative SDS-polyacrylamide gels into a 300-microliter recovery volume. After an 8-h elution period, recovery ranged from 80 (193 kDa) to 97% (33 kDa). The "losses" were generally due to proteins still remaining in the gel slice. All of the eluted proteins tested in a dot-blot assay proved to be antigenically active. The advantages of the device described here are easy handling (insertion of membranes, open system), quantitative recovery, and high reproducibility of the elution results.

Complete DNA sequence of lymphotropic papovavirus: prototype of a new species of the polyomavirus genus.

Lymphotropic papovavirus (LPV) is a new member of the polyomavirus genus. Its host range in vitro is restricted to transformed cells of B-lymphocyte origin. Here the complete 5270-bp DNA sequence of LPV is presented. The LPV early region can encode a large T and a small t antigen but no middle T antigen and the late region can encode the three structural proteins VP1, VP2, and VP3. Based on sequence conservation of shared proteins LPV is equally related to both mouse polyomavirus (Py) and simian virus 40 (SV40) and represents a new distinct species of the polyomavirus genus. Sequence comparisons of LPV, SV40, and Py point out essential conserved sequence features of the polyomavirus genus more clearly than the comparison of only SV40 and Py. The most conserved proteins are VP1 with 42% and large T antigen with 28% of the amino acids conserved among the three viruses. Although least conserved the noncoding DNA sequences of LPV show significant homologies both to SV40 and Py (origin of viral DNA replication and putative early promoter). A 63-bp tandem repeat at the late side of the replication origin possibly represents a LPV enhancer element.