How to detect Chlamydia trachomatis in males?

Much is known about the role of Chlamydia trachomatis in female infertility, although the same cannot be said about the organism's role in male infertility. Recently a number of researchers have provided a possible explanation of the pathogenesis of C trachomatis in male infertility and have suggested further studies. Unfortunately, current screening recommendations for C trachomatis in an infertile couple are vague and unhelpful, and many do not even mention this type of screening in the male. To enable any progress to be made in this field, it is essential that investigators know how best to detect C trachomatis, especially in the male. It is important, therefore, to know which specimen is best for C trachomatis detection, with respective strengths and weaknesses of each specimen. Similarly, it is equally important to have knowledge of which test is appropriate for the type of specimen being examined. First void urine is currently the specimen of choice for the routine detection of C trachomatis in males. Moreover, the best detection protocols in the developed world are based on molecular diagnosis of first void urine. These methods provide the best combination of sensitivity and specificity that is currently available on a clinical sample that can be self-taken. Interestingly, because semen is routinely collected for analysis in men of infertile couples, it has been suggested that protocols be developed for the optimal detection of C trachomatis in this specimen. Semen might provide additional information on infection of the upper genital tract, which may not be detected in first void urine. Finally, the importance of comparing tests for C trachomatis detection in updating our knowledge has been highlighted by the inability of some molecular methods to detect the new variant strain of C trachomatis.

Innate immunity in the male genital tract: Chlamydia trachomatis induces keratinocyte-derived chemokine production in prostate, seminal vesicle and epididymis/vas deferens primary cultures.

Chlamydia trachomatis is an intracellular pathogen that infects mucosal epithelial cells, causing persistent infections. Although chronic inflammation is a hallmark of chlamydial disease, the proinflammatory mechanisms involved are poorly understood. Little is known about how innate immunity in the male genital tract (MGT) responds to C. trachomatis. Toll-like receptors (TLRs) are a family of receptors of the innate immunity that recognize different pathogen-associated molecular patterns (PAMPs) present in bacteria, viruses, yeasts and parasites. The study of TLR expression in the MGT has been poorly investigated. The aim of this work was to investigate the keratinocyte-derived chemokine (KC) response of MGT primary cultures from C57BL/6 mice to C. trachomatis and different PAMPs. KC production by prostate, seminal vesicle and epididymis/vas deferens cell cultures was determined by ELISA in culture supernatants. TLR2, 3, 4 and 9 agonists induced the production of KC by all MGT primary cultures assayed. In addition, we analysed the host response against C. trachomatis and Chlamydia muridarum. Chlamydial LPS (cLPS) as well as C. trachomatis and C. muridarum infection induced KC secretion by all MGT cell cultures analysed. Differences in KC levels were observed between cultures, suggesting specific sensitivity against pathogens among MGT tissues. Chemokine secretion was observed after stimulation of seminal vesicle cells with TLR agonists, cLPS and C. trachomatis. To our knowledge, this is the first report showing KC production by seminal vesicle cells after stimulation with TLR ligands, C. trachomatis or C. muridarum antigens. These results indicate that different receptors of the innate immunity are present in the MGT. Understanding specific immune responses, both innate and adaptive, against chlamydial infections, mounted in each tissue of the MGT, will be crucial to design new therapeutic approaches where innate and/or adaptive immunity would be targeted.

Analysis of modulated gene expression in a model of Interferon-gamma-induced persistence of Chlamydia trachomatis in HEp-2 cells.

BACKGROUND: Chlamydia trachomatis is an important pathogen, being the commonest sexually transmitted bacterial disease in the Western world and is also implicated in a number of acute and chronic diseases. Persistent infections of C. trachomatis are particularly associated with chronic infections, which although eliciting an immune response, result in tissue damage leading to complications such as pelvic inflammatory disease. Interferon (IFN)-gamma is known to induce persistent infections of C. trachomatis both in vitro and in vivo. METHODS: A model of IFN-gamma-induced persistence containing aberrant inclusions of C. trachomatis was developed in the HEp-2 cell line. Morphological changes to inclusions were assessed by fluorescence immunocytochemistry and transcript levels determined by Real-Time RT-PCR. To assess infectivity of C. trachomatis in an IFN-gamma-induced persistent state, cultures containing aberrant inclusions were inoculated onto fresh HEp-2 monolayers. RESULTS: IFN-gamma induced aberrant inclusion formation at 0.01 ng/ml. Doses from 0.05 to 100 ng/ml did not significantly increase numbers of aberrant inclusions, and some normal inclusions were observed at the highest dose of IFN-gamma. Transfer of IFN-gamma-treated C. trachomatis onto fresh cultures confirmed the infectivity of these cultures. Real-Time RT-PCR identified apparent increased expression of the C. trachomatis heat-shock response genes ct604 and ct755 at 96-h post-infection. However comparisons with control cultures suggest that this more likely reflects a failure to down regulate gene expression as observed in untreated cultures. CONCLUSIONS: These data show that whereas IFN-gamma induces aberrant inclusion formation, many normal inclusions are still observed at high doses of IFN-gamma, and that the infectivity of such cultures is presumably from these. Transcriptional changes observed in response to IFN-gamma suggest a failure of the C. trachomatis life cycle in response to IFN-gamma, however IFN-gamma-induced transcriptional changes may be masked by the presence of normal inclusions. The implications of these observations in relation to models of persistence of C. trachomatis are discussed.

Is the role of Chlamydia trachomatis underestimated in patients with suspected reactive arthritis?

Reactive arthritis is usually caused by bacteria of either the enteric or genital tracts. In the genital tract, Chlamydia trachomatis is perhaps the only aetiological agent. In Iran, newer evidence suggests that as C. trachomatis is more commonly found in the general population than was previously believed, its role in reactive arthritis may well be currently overlooked. In this review, as well as emphasizing the potential role of C. trachomatis in reactive arthritis in patients from developing countries, we also make recommendations for further clinical studies to determine its prevalence. Moreover, we also stress the need for standardization of new testing methodologies for C. trachomatis, including the use of new commercial systems in an attempt to determine a truer picture of chlamydial infection in reactive arthritis.

Role of activins and inducible nitric oxide in the pathogenesis of ectopic pregnancy in patients with or without Chlamydia trachomatis infection.

Chlamydia trachomatis infection can lead to pelvic inflammatory disease, ectopic pregnancy (EP), infertility, and chronic pelvic pain in women. Activins and inducible nitric oxide synthase (iNOS) are produced by the human fallopian tube, and we speculate that tubal activins and iNOS may be involved in the immune response to C. trachomatis in humans and their pathological alteration may result in tubal pathology and the development of EP. Blood and fallopian tubes were collected from 14 women with EP. Sera were analyzed by enzyme-linked immunosorbent assay to detect antibodies against chlamydial heat shock protein 60 (chsp60) and the major outer membrane protein of C. trachomatis. Confirmation of C. trachomatis serology was made using the microimmunofluorescence test. The patients were classified into three groups according to their serological results, and immunohistochemistry and quantitative reverse transcription-PCR were performed to investigate the expression of candidate molecules by tubal epithelial cells among the three groups. This is the first study to show an increase in the expression of activin betaA subunit, type II receptors, follistatin, and iNOS within the human fallopian tube of EP patients who were serologically positive for C. trachomatis. A similar expression profile was observed in the fallopian tubes with detectable antibodies only against chsp60. These results were shown at the mRNA and protein levels. We suggest that tubal activin A, its type II receptors, follistatin, and NO could be involved in the microbial-mediated immune response within the fallopian tube, and their pathological expression may lead to tubal damage and the development of EP.

Real-time polymerase chain reaction shows that density centrifugation does not always remove Chlamydia trachomatis from human semen.

OBJECTIVE: To evaluate the efficiency of sperm washing procedures to remove Chlamydia trachomatis from semen both in clinical samples and experimental inoculations. DESIGN: Laboratory-based study. SETTING: Research laboratory in a university hospital. PATIENT(S): One hundred men attending for diagnostic semen analysis as part of infertility investigations and three sperm donors providing ejaculates for research purposes. MAIN OUTCOME MEASURE(S): Number of DNA copies of C. trachomatis, infectivity in an HeLa cell monolayer, and immunofluorescence. RESULT(S): Of the 100 semen samples examined, 13 contained detectable levels of C. trachomatis DNA (675-15,920 copies/mL) and in only 7 was this completely removed after sperm washing. In the remaining six DNA-positive samples, the number of copies in the postwash preparation ranged from 36-455 per mL. Experimental inoculations found that postwash preparations containing C. trachomatis DNA as low as 61 copies/mL were able to establish an infection in vitro. CONCLUSION(S): Undiagnosed C. trachomatis infections in men attending for assisted conception could potentially lead to infection or contamination of the IVF culture system as sperm washing methods are not 100% effective.

Differential glycosaminoglycan binding of Chlamydia trachomatis OmcB protein from serovars E and LGV.

We recently showed that OmcB protein from Chlamydia trachomatis serovar LGV1 functions as an adhesin. In this study, we produced Escherichia coli expressing OmcB from serovar E and compared this OmcB to OmcB from serovar LGV1. Infectivity inhibition assays carried out with serovars LGV1 and E of C. trachomatis in the presence of recombinant OmcB showed considerable (approximately 60%) inhibition of infectivity. In the presence of heparan sulphate, there was significant inhibition (68%) of adherence of E. coli expressing OmcB from serovar LGV1 only. In a further experiment, recombinant OmcB from serovar LGV1 showed minimal binding to glycosaminoglycan (GAG)-deficient cells, whilst to the same cells, recombinant OmcB from serovar E showed binding equal to that to the wild-type cells. Our experiments strongly suggest that OmcB from serovar E, in contrast to that from serovar LGV1, is not binding to host cells through a GAG-dependent mechanism.

Is lipopolysaccharide a factor in infectivity of Chlamydia trachomatis?

Lipopolysaccharide (LPS) is a major surface component of Chlamydia trachomatis, as with all Gram-negative bacteria. The effect of C. trachomatis LPS on C. trachomatis infectivity of human epithelial cells was investigated. C. trachomatis LPS and C. trachomatis LPS antibody significantly reduced infectivity, mostly in a dose-dependent manner. As the structure of LPS in C. trachomatis is simple and consists only of lipid A and 3-deoxy-D-manno-octulosonic acid (Kdo), we investigated whether lipid A or Kdo was inhibitory to chlamydial infectivity. Polymyxin B, as a lipid A inhibitor, and Kdo considerably reduced C. trachomatis infectivity. With all the LPS inhibitors used, there was greater inhibition against serovar E than serovar LGV. These results suggest a role for LPS in chlamydial infectivity. Elucidation of how LPS acts in infectivity and identification of host-cell receptors would help in understanding pathogenicity.

Activities of the frog skin peptide, ascaphin-8 and its lysine-substituted analogs against clinical isolates of extended-spectrum beta-lactamase (ESBL) producing bacteria.

Extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria are becoming increasingly prevalent and their antibiotic resistance necessitates novel therapeutic intervention. Ascaphin-8 is a cationic alpha-helical peptide that shows broad-spectrum antibacterial activity but is also toxic to human erythrocytes (LC(50)= 55 microM). This study assesses the activity of ascaphin-8, and a series of l-lysine-substituted analogs, against a range of clinical isolates of ESBL-producing bacteria. All ESBL-producing Escherichia coli (MIC=1.5-6 microM) and Klebsiella pneumoniae (MIC=12.5-25 microM) strains tested were susceptible to ascaphin-8, as well as a group of miscellaneous ESBL-producing strains (Citrobacter, Salmonella, Serratia, Shigella spp.) (MIC< or = 25 microM). The Lys4- and Lys8-substituted analogs were generally the most potent against bacteria but showed the highest hemolytic activity. However, the Lys10, Lys14, and Lys18 analogs also displayed potent antibacterial activity while showing very low hemolytic activity (LC50> 500 microM). An unexpected finding was the susceptibility of ESBL-producing Proteus mirabilis strains to ascaphin-8 (MIC=12.5-25 microM) and its Lys4-substituted analog (MIC= 6 microM), although non-ESBL isolates of this organism were resistant to these peptides (MIC> 100 microM).

Interaction of Chlamydia trachomatis serovar E with male genital tract epithelium results in secretion of proinflammatory cytokines.

Although much has been reported on the in vitro interaction of Chlamydia trachomatis with cells derived from the female genital tract, little is known of its interaction with male genital tract epithelium. The aim of this work was to investigate the effect of C. trachomatis serovar E on immortalized normal human urethral epithelial cells and on immortalized normal adult human prostate epithelial cells with regard to chlamydial growth and secretion of cytokines. After infection, these epithelial cells were assessed for their support of chlamydial growth in comparison with HeLa cells, and cytokine levels in cell culture supernatants were determined by ELISA. Although the male-derived epithelial cells supported growth of chlamydiae, the best growth was seen in HeLa cells. In contrast to prostate epithelial cells, the urethral epithelial cells released much larger quantities of interleukin 1alpha (IL-1alpha) following infection, whereas both IL-6 and IL-8 were produced in larger quantities by infected prostate cells. At 7 days post-infection, HeLa cells consistently produced large quantities of all three cytokines. In conclusion, the male-derived cell lines were shown to support the invasion of C. trachomatis and initiate a proinflammatory response to infection. From in vitro studies the suggestion that high levels of IL-6 could be a possible marker for chlamydial prostatitis is confirmed. Although not as marked a change, it is also suggested that higher IL-8 levels could be associated more with infection of the prostate than the urethra. Differential cytokine production by different male-derived epithelial cells could help determine the site of chlamydial infection and help in the study of pathogenesis.

Apoptotic effect on HeLa cells produced by Chlamydia trachomatis-LPS

La interacción entre el lipopolisacárido (LPS) de Chlamydia trachomatis y las células de mamíferos permanece sin ser dilucidado. Chlamydia trachomatis es una bacteria intracelular responsable de diversas enfermedades en los humanos y animales. En este trabajo mediante el aislamiento del lipopolisacárido de dos serovares de Chlamydia trachomatis (LGV1-LGV2) y usando una coloración Supravital fluorescente (Hoechst 33258) fue posible investigar la respuesta de las células HeLa. El efecto apoptótico que sufren este tipo de células fue visible cuando fueron expuestas a dicho LPS en concentraciones iguales o mayores que 0,5 µg/mL por un periodo de 48 horas, sin embargo se observó la falta de repuesta celular en su ausencia o en presencia de LPS de otras bacterias. Adicionalmente, el uso en iguales condiciones de polimyxina B conocido como un neutralizador de la acción del LPS demostró una disminución del efecto apoptótico en dichas células, indicando que la respuesta celular observada fue producida por C. trachomatis-LPS. Los resultados de este trabajo le dan fuerza a la teoría de que el LPS de C. trachomatis pudiera ser el responsable del efecto tóxico que se observa sobre las células cervicales infectadas con esta bacteria intracelular.

Chlamydia trachomatis OmcB protein is a surface-exposed glycosaminoglycan-dependent adhesin.

The OmcB protein of Chlamydia trachomatis is a cysteine-rich outer membrane polypeptide with important functional, structural and antigenic properties. The entire gene encoding the OmcB protein from C. trachomatis serovar LGV1 was cloned and expressed in Escherichia coli and the full-length protein used to raise polyclonal antibodies. Recombinant OmcB was used to show that OmcB is a surface-exposed protein that functions as a chlamydial adhesin. Infectivity inhibition assays carried out using HeLa cells with serovar LGV1 in the presence of purified anti-OmcB serum showed inhibition of infectivity, suggesting that some of the OmcB was surface exposed. Moreover, using recombinant OmcB in infectivity inhibition assays resulted in 70% inhibition of infectivity, confirming that OmcB plays a role as an adhesin in C. trachomatis. Furthermore, recombinant OmcB protein bound to the surface of HeLa and Hec1B cells, but binding to glycosaminoglycan (GAG)-deficient cells (pgsA-745 and pgsD-677) was markedly reduced, indicating that OmcB binds to GAG-like receptors on host cells.

Chlamydia trachomatis prevalence in Iranian women attending obstetrics and gynaecology clinics.

This study was designed to estimate the prevalence of Chlamydia infection in women attending Obstetrics and Gynaecology clinics in Tehran, during May 2003 to October 2003. Women attending Obstetrics and Gynaecology clinics aged 15-42 were recruited by Sequential Random Sampling. Those who had not passed urine in the last hour were eligible. Informed consent was obtained and a questionnaire completed after being interviewed by a midwife. First void urine was collected and after DNA extraction from urine specimen, PCR tests were performed; urine DNA samples were tested by strand displacement amplification (SDA) for Chlamydia confirmation. 12.6% (133/1052) tested positive for Chlamydia by PCR. Of these PCR positive samples, 86 were available for re-testing by SDA and 67 were positive giving a correlation between the tests of 78%. This gave an overall true prevalence of 6.4% which is however, underestimated. No statistical differences were seen between patient age groups, details of personal and reproductive history and combined PCR and SDA positivity for C. trachomatis. A 12.6% prevalence of Chlamydia trachomatis was found by PCR testing which is cost effective to screen and treat. Despite limitations in re-testing PCR-positive samples by SDA, a 78% correlation between tests confirms a high prevalence of C. trachomatis. Non-invasive screening of women was therefore a success in this group of patients. As this was the first time that more sensitive molecular methods were used for detection of C. trachomatis, prevalence in such a big sample size, the results are considerable. However, we suggest further such testing.

Spermicidal activity of bacterial lipopolysaccharide is only partly due to lipid A.

We have previously shown that co-incubation of Chlamydia trachomatis lipopolysaccharide (LPS) leads to premature sperm death by an apoptosis-like mechanism. It was always assumed that lipid A is the toxic component of LPS. Here we investigate the possible involvement of 3-deoxy-D-manno-octulosonic acid (Kdo), which is an additional component of the LPS in C. trachomatis. Highly motile preparations of sperm from normozoospermic patients were incubated for 6 hours with commercial sources of lipid A and Kdo. Conventional lipid A inhibitors, polymyxin B (PMB) and anti-CD14 monoclonal antibody (mAb) were used to test the ability of both lipid A and Kdo to induce an apoptotic-like response in mature sperm. Flow cytometry was used to determine apoptosis by the expression of annexin V. Caspase activity was also measured by fluorometry and by the use of a pan-caspase inhibitor and caspase-3 inhibitor. Both lipid A and Kdo at 50 micro g/mL caused significant mortality of sperm. However, although PMB and anti-CD14 mAb were inhibitory to the activity of lipid A on sperm, no such effect was seen against Kdo. In the presence of either lipid A or Kdo, sperm death was caused by an apoptotic-like effect that was caspase mediated. We conclude that Kdo shares its spermicidal properties with lipid A and seems to kill sperm in a similar manner. These results provide an explanation for higher than expected levels of spermicidal activity of LPS that are not caused by lipid A.

Effect of storage temperature on survival of Chlamydia trachomatis after lyophilization.

Lyophilized preparations of Chlamydia trachomatis were made to investigate how well they would survive storage at four relevant incubation temperatures for 1 week and 1 month. Good viability was maintained by storage at either 4 degrees C or 20 degrees C for 1 week. If the ambient temperature is not too high, short-term transportation of C. trachomatis is achievable through lyophilization.

Can Chlamydia trachomatis directly damage your sperm?

Although Chlamydia trachomatis causes symptomatic infection in the lower genital tract of approximately 50% of men, its role in the upper genital tract is less well known. Moreover, for a number of reasons, mostly based on methodological aspects, the impact of chlamydia on semen quality is controversial. Overall, in-vivo studies of C trachomatis in men have provided conflicting evidence as to whether it is associated with reduced fertility. By contrast, in-vitro studies show that co-incubation of spermatozoa with chlamydia causes a significant decline in numbers of motile sperm and results in premature sperm death. Since evidence suggests that chlamydial lipopolysaccharide is the principal factor leading to sperm apoptosis, a new line of inquiry would be to measure the levels of lipopolysaccharide in semen and relate these to parameters of semen quality, including that of sperm function. If these new lines of inquiry are proven, this could lead to potentially novel approaches in the treatment of infertile men.

Lipopolysaccharides of Bacteroides fragilis, Chlamydia trachomatis and Pseudomonas aeruginosa signal via toll-like receptor 2.

Recognition of bacterial lipopolysaccharide (LPS) is critical in the host defence against Gram-negative infection. While enterobacterial LPS signals via Toll-like receptor 4 (TLR4), it has recently been reported that the LPS of Leptospira interrogans, Legionella pneumophila, Rhizobium species Sin-1 and at least one strain of Porphyromonas gingivalis are capable of signalling via TLR2. Using a TLR transfection assay and measurement of an NF-kappaB-sensitive promoter region, the results show that the LPS of Bacteroides fragilis NCTC-9343, Chlamydia trachomatis LGV-1 and Pseudomonas aeruginosa PAC-611 also signal via TLR2 and it is pointed out that all TLR2-signalling LPS discovered to date demonstrate relatively weak endotoxicity in some models and structural features distinct from those LPS shown to signal via TLR4.

Chlorate: a reversible inhibitor of proteoglycan sulphation in Chlamydia trachomatis-infected cells.

Sulphated glycosaminoglycans, such as heparan sulphate, have been shown to be essential for the infectivity of many organisms. The aims of this study were to verify the role of sulphated glycosaminoglycans in chlamydial infection and to investigate whether they are present on chlamydia or chlamydial host cells. The effect of undersulphation of host cells and chlamydial elementary bodies was examined using sodium chlorate. Also studied was whether any inhibitory effect was reversible. The results strongly suggest that Chlamydia trachomatis does not produce heparan sulphate and that heparan sulphate of the host cell is necessary and sufficient to mediate chlamydial infection. The essential role played by the sulphate constituents of the host-cell glycosaminoglycan in the infectivity of LGV serovars, and to a lesser extent of serovar E, was also confirmed.

Semen quality of men with asymptomatic chlamydial infection.

We have shown previously that the in vitro exposure of spermatozoa to elementary bodies (EBs) of Chlamydia trachomatis can lead to sperm death over a number of hours of incubation. As such, we have hypothesized that the ejaculates of men with a chlamydial infection could contain increased numbers of nonmotile (dead) spermatozoa if they are exposed to EBs prior to ejaculation. To test this hypothesis, the ejaculates of 642 men undergoing diagnostic semen analysis as part of ongoing infertility investigations with their partner were examined. All men were without symptoms of genitourinary infections and semen analysis was performed according to World Health Organisation (WHO) 1999 methods after a 3-5 day abstinence period. In addition to semen analysis, nested plasmid polymerase chain reaction (PCR) was undertaken on the ejaculate to detect the presence of C trachomatis DNA. A total of 31 semen specimens (4.9%) were found to be positive, and in 28 of these, the diagnosis was confirmed using the ligase chain reaction (LCR). Men whose ejaculates were PCR positive for chlamydial DNA had a significantly (P <.05) higher mean concentration of leukocytes (1.71 +/- 2.20 x 10(6) per mL) and a higher mean ejaculate volume (3.45 +/- 1.52 mL) than in those whose ejaculates were PCR negative (leukocyte concentration: 0.67 +/- 2.59 x 10(6) per mL; volume 2.93 +/- 1.38 mL). Leukocytospermia was twice as common in men that were PCR positive for chlamydial DNA (P <.05) but it was not always associated with the presence of chlamydial DNA in semen. However, there was no difference in the mean percent motility between the 2 groups and the proportion of asthenozoospermia also did not differ. Because these results do not confirm the hypothesis proposed from our in vitro experiments, further work needs to be undertaken to understand whether human spermatozoa are actually exposed to elementary bodies of C trachomatis in an infected individual prior to ejaculation.