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Chlamydial infection of the lower genital tract usually spreads to the upper genital tract and is then responsible for more serious consequences, such as infertility, ectopic pregnancy, pelvic pain, and pelvic inflammatory disease. Genital infection with Chlamydia trachomatis and the resulting cytokine response largely determines the outcome of infection and disease. To date, studies showing comparative effects of azithromycin and doxycycline treatment for C. trachomatis infection in women with reproductive sequelae like infertility and their effect on immune molecules like cytokines are lacking. Hence, our objective was to study the effect of azithromycin and doxycycline in vitro on cytokines in cells from C. trachomatis-positive fertile and infertile women as well as their efficacy in C. trachomatis infection. Fertile and infertile women with primary and recurrent C. trachomatis infection attending the gynecology outpatient department of Safdarjung Hospital, New Delhi, India, were enrolled. Enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction was performed for evaluating cytokines in cells stimulated with chlamydial elementary bodies (EBs) in the presence and absence of antibiotics (azithromycin and doxycycline). C. trachomatis-infected women were also followed up to assess the efficacy of azithromycin and doxycycline. We observed inhibition of cytokines (interleukin [IL]-1beta (β), IL-6, IL-8, IL-10, and tumor necrosis factor-alpha) in the presence of azithromycin in EB-stimulated cells from both fertile and infertile women with primary and recurrent C. trachomatis infection. However, in presence of doxycycline, inhibition of cytokines (IL-1β and IL-6) was only observed in stimulated cells from fertile women with primary C. trachomatis infection. The clinical efficacy of azithromycin was also better than doxycycline in recurrent C. trachomatis infection in women with complications such as infertility. Overall, this study suggests that azithromycin treatment with broader immunomodulatory effects may be preferable to doxycycline for the treatment of recurrent C. trachomatis infection associated with infertility.

Epidemiological and animal model studies suggest that sequelae of genital Chlamydia trachomatis infection are more often associated with second or subsequent infections than with initial infection. Further, in order to establish an acute or long-term persistent infection, C. trachomatis develops several strategies to circumvent host immune responses. Hence, resolution of the C. trachomatis infection may require modulation of host factors especially during persistent or chronic infection. Moreover, azithromycin treatment has been reported to possess anti-inflammatory properties but its mechanism of action is still not elucidated. Therefore, in order to better understand the effect of azithromycin in chronic conditions, our aim was to study changes in expression of key genes associated with inflammatory cytokines and receptors, mitogen-activated protein kinase (MAPK) signaling pathway, and apoptosis pathway before and after therapy with azithromycin in infertile women with recurrent C. trachomatis infection. Real-time polymerase chain reaction was performed to study inflammatory cytokines and receptors, MAPK signaling pathway, and apoptosis pathway before and after therapy with azithromycin in infertile women with recurrent C. trachomatis infection. Further, effect of azithromycin on activation of extracellular signal-regulated kinase was studied in epithelial cells by western blotting. Chemokine (C-C motif) ligand 2 (CCL2), CCL5, chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL5, CXCL9, interleukin-1B (IL-1B), IL-8, baculoviral IAP repeat-containing 3 (BIRC3), myeloid cell leukemia sequence 1 (MCL1), and MAPK1 were downregualted after azithromycin treatment. In addition, phosphorylation of extracellular signal-regulated kinase was inhibited after azithromycin treatment in epithelial cells obtained from women with recurrent infection. Hence, our data suggest that azithromycin with its properties apart from antibacterial activity may contribute to its therapeutic potential in treatment of chronic recurrent infection in infertile women.

To investigate whether IL-17A (IL-17) and IL-22 are produced in response to Chlamydia trachomatis infection, the cervical washes of 27 women with C. trachomatis infection and 17 C. trachomatis negative controls were collected. The levels of cytokines were determined in the cervical wash and in the supernatant of cervical and systemic cell cultures upon C. trachomatis antigen stimulation. C. trachomatis infection appeared to activate local IL-17 and IL-22 production more efficiently than IFN-γ production. In the cervical wash of infected women, median concentrations of IL-17 and -22 were 5- and 3-fold higher, respectively, than in negative controls. The spontaneous intracellular expression of these cytokines was analysed by flow cytometry in blood and cervical cells and 26% of cervical mononuclear cells from infected women were shown to produce IL-22 and 12% to coproduce IL-17 and IL-22. In addition, it was demonstrated that 20-25% of IL-22 producing and IL-17-IL-22 coproducing cervical CD4+ T cells expressed the mucosal homing receptor CCR6. These results suggest that CCR6 is involved in the migration of these cells to the cervix and that IL-17 and IL-22 might play a role in the immune response at the site of C. trachomatis infection., (© 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

Chlamydiae are obligate intracellular bacteria that infect human epithelial cells. It has been reported that Chlamydia trachomatis, induces apoptosis in epithelial cells, however, the molecular mechanisms responsible for host cell death especially in primary epithelial cells remained largely unknown as most of the studies are in cell line like HeLa. In this study we demonstrated that C. trachomatis induces apoptosis signaling pathway and apoptosis in primary cervical epithelial cells in a time and dose dependent manner. Live cervical epithelial cells were isolated from endocervical cells and induction was done with chlamydial EBs. Our results demonstrated that apoptosis in infected epithelial cells was associated with an increased activity of caspase 8; however, caspase 9 was activated to a lesser extent. Analysis of apoptosis pathway revealed that expression level of McL-1, Bcl-2, CASP8, and TRADD genes were found to be significantly upregulated (P < 0.01), where as levels of Caspase 1, Caspase 10 and BRIC2 were found to be significantly downregulated (p < 0.01). Our results showed that Chlamydia induces apoptosis and caspase activation in epithelial cells through caspase 8, with an increased expression of the McL-1, which confers a block at the mitochondrial level.

Background: Recurrent genital Chlamydia trachomatis infection often results in serious sequelae and has a major impact on reproductive health., Materials and Methods: Recurrent infections were determined in symptomatic female patients. In vitro susceptibility assay was performed for azithromycin and doxycycline using the cell culture technique against 21 clinical isolates obtained from C. trachomatis-positive patients including those who were recurrently infected., Results: Thirteen isolates (61.9%) were found to be susceptible to azithromycin and doxycycline with minimum inhibitory concentration (MIC) values ≤0.125 and ≤0.25 μg/ml, respectively. Eight isolates (38%) were found to be less susceptible to the drugs. Two of them had MICs of 8 μg/ml for both the drugs and could not be completely eradicated as observed by minimum bactericidal concentration assay., Conclusions: Decreased antibiotic susceptibility to the current first-line drugs (azithromycin and doxycycline) for chlamydial infection treatment was observed in isolates obtained from recurrently infected patients., (Copyright © 2010 S. Karger AG, Basel.)

Sexually transmitted Chlamydia trachomatis infection is an important public health concern with major adverse effects on female reproductive tract health and function. The magnitude of reproductive morbidity associated with sexually transmitted C. trachomatis infection is enormous, however to date no prophylactic vaccine is available. In part this is due to the lack of information on the mucosal immunobiology of the host-pathogen interaction and correlates of protective immunity during genital C. trachomatis infection. In this review, we focus on current knowledge of mucosal innate and adaptive immune responses in the female genital tract during C. trachomatis infection, which will eventually help in the development of a vaccine for prevention of chlamydial infection.

Background: Chlamydial Inclusion membrane proteins (Incs), are involved in biochemical interactions with host cells and infecting Chlamydiae. We have previously reported the role of two Chlamydia trachomatis (CT) Incs, namely IncB and IncC in generating host immunity in CT infected women. Emerging data shows involvement of Inc stimulated CD4 positive T cells in aiding host immunity in infected fertile and infertile women through the secretion of interferon gamma. However the lack of data on the intra-cytokine interplay to these Incs in infected cell milieu prompted us to investigate further., Methods: A total of 14 CT-positive fertile, 18 CT-positive infertile women and 25 uninfected controls were enrolled in this study. CD8 depleted, CD4 enriched cervical cells were isolated and upon stimulation with IncB and IncC, modulation of cytokines (Interleukin (IL)-1 Beta, IL-4, IL-5, IL-6, IL-10, Interferon-gamma, IL-12, IL-23, Tumor Necrosis Factor-alpha and Granulocyte macrophage colony-stimulating factor (GM-CSF) and T cell lineage regulating transcription factors T-Bet and GATA3 was determined by real-time reverse-transcriptase (RT)-PCR and ELISA., Results: Significant higher expression (P < 0.05) of Interferon-gamma, IL-12, IL-23 and GM-CSF were found in Inc-stimulated CD4 enriched cervical cells of CT-positive fertile women and contrastingly high IL-1 Beta, IL-4, IL-5, IL-6 and IL-10 levels were found in CT-positive infertile women. Positive correlation (P < 0.05) was found between Interferon-gamma and T-Bet levels in CT-positive fertile women and IL-4 mRNA and GATA3 levels in CT-positive infertile patients upon IncB and IncC stimulation., Conclusion: Overall our data shows that CT IncB and IncC are able to upregulate expression of cytokines, namely interferon-gamma, IL-12, IL-23 and GM-CSF in CT-positive fertile women while expression of IL-1 Beta, IL-4, IL-5, IL-6 and IL-10 were upregulated in CT-positive infertile women. Our study also suggests that Incs are able to modulate expression of T cell lineage determinants indicating their involvement in regulation of immune cells.

Background: Proteins in the inclusion membrane of Chlamydia trachomatis (CT) have been anticipated to play pivotal roles in the molecular and cellular interactions between the pathogen and host. However, there is lack of data on host immunity with respect to antibody responses against chlamydial inclusion proteins., Methodology: We used full-length fusion proteins for CT inclusion membrane proteins B and C (IncB and IncC respectively), two early-infection phase proteins, to study their role in antibody generation during human infection., Results: Three hundred and fifty-five women (aged 22-36 years) attending the Gynaecology outpatient department, Safdarjang Hospital, New Delhi, India were enrolled in this hospital ethical committee approved study. Out of these, 108 were diagnosed to be cervical CT-positive. Of these 108 patients, 67 (62.03%) showed ELISA positivity for IncB IgG, and 64 (59.25%) for IncC IgG. There was a positive correlation between antibody titres against IncB and IncC and with antibodies against CT major outer membrane protein (MOMP) in CT-positive sera. Our data also showed a positive association between antibody titres against IncB and IncC in patients with cervicitis and pelvic inflammatory disease (PID). Significantly high antibody titres were detected in cervicitis cases compared with PID. There were significantly higher levels of serum cytokines (TNF-alpha, IFN-gamma and IL-12) in Inc-positive cervicitis cases than in PID cases. In addition, our study also showed higher IncB and IncC IgG(2) titres in comparison to respective IgG1, IgG3 and IgG4 titres in CT-positive sera., Conclusion: Our data suggested that antibodies against CT IncB and IncC were prevalent in CT-positive women diagnosed with cervicitis or PID.

Background: With an increase in the number of putative inclusion membrane proteins (incs) in chlamydial genomes, there is a need for understanding their contribution in host-pathogen interactions. Thus in this study we determined the host mucosal and peripheral immune responses to incs (IncB and IncC) of Chlamydia trachomatis (CT)., Methods: Female patients (n = 296) attending the gynaecology out patient department of Safdarjung hospital, New Delhi were enrolled for the study and were clinically characterized into two groups; CT-positive fertile women (n = 38) and CT-positive women with fertility disorders (n = 29). Uninfected healthy fertile women were enrolled as controls (n = 31). Gene specific PCRs were used for detection of incB and incC genes in endocervical samples of CT-positive patients. ELISA and Western blot assay were used for detection of IgA and IgG antibodies to IncB and IncC in cervical washes and sera. Effect of IncB and IncC stimulation of cervical cells and PBMCs on cellular proliferation and cytotoxity was determined using MTT assay and Lactate dehydrogenase (LDH)-cytotoxicity assay respectively. Modulation of cytokines (Interleukin (IL)-1 Beta, IL-4, IL-5, IL-6, IL-10, Interferon-gamma, IL-12, Tumor Necrosis Factor-alpha and Granulocyte macrophage colony-stimulating factor (GM-CSF)) in cervical cells and PBMCs upon stimulation with IncB and IncC was determined by real-time reverse-transcriptase (RT)-PCR and ELISA. Further, CD4 positive T cells were purified from cervical cells and peripheral blood mononuclear cells (PBMCs) and secreted cytokines (Interferon-gamma and IL-4) were evaluated by ELISPOT and real-time RT-PCR., Results: Using MTT assay, significantly high proliferative responses (P < 0.05) were observed in inc-stimulated cervical cells and PBMCs from CT-positive fertile women compared to CT-positive women with fertility disorders and controls. Interferon-gamma, IL-12 and GM-CSF were found to be elevated in inc-stimulated cervical cells and PBMCs of CT-positive fertile women compared to CT-positive women with fertility disorders and controls (P < 0.05). In contrast, IL-1 Beta, IL-4, IL-5, IL-6 and IL-10 levels were found to be higher in CT-positive women with fertility disorders compared to CT-positive fertile women and controls (P < 0.05). Interferon-gamma secreting cells and mRNA expression in inc-stimulated cervical and peripheral CD4 positive T cells were significantly higher (P < 0.05) in CT positive fertile women compared to CT-positive women with fertility disorders., Conclusion: Our data overall suggests that CT incs, IncB and IncC modulate host immune responses and may have a role in protection/pathogenesis of genital chlamydial infection in women.

Little is known about genital mucosal immune response to chlamydial infection in women with or without sequelae (Chlamydia positive women with or without fertility disorders as infertility and multiple spontaneous abortions). Cervical lymphocytes were stimulated with chlamydial EBs and cytokine secretion was determined by ELISA, RT-PCR and ELISPOT assays. Stimulated cervical cells from women with fertility disorders (FD) secrete significantly (P<0.05) higher levels of IL-1beta, IL-6, IL-8 and IL-10 and cells from fertile women secrete significantly higher levels of IL-12 and IFN-gamma compared to other groups. RT-PCR analysis showed similar results for IFN-gamma and IL-12. For IL-10 and IL-4, mRNA expression levels were significantly higher (P<0.05) in cells obtained from women with FD compared to other groups. Results for ELISPOT assay were similar as those of RT-PCR. The results suggest that cytokine secretion profile of cervical cells may decide whether infection does not hamper fertility or will develop fertility disorder.

The regulation of immune response and chlamydial infectious load in the cervix of human females is largely unknown. Infectious load in terms of inclusion-forming units (IFUs) was determined by quantitative cultures in Chlamydia-positive women, in asymptomatic women, women with mucopurulent cervicitis (MPC) and women with fertility disorders (FD). CD4(+), CD8(+), CD14(+) cells, myeloid and plasmacytoid dendritic cells (mDCs and pDCs) in the cervix were quantified by flow cytometry. Cervical cytokines, levels of beta-estradiol and C-reactive protein (CRP) in serum and cervical immunoglobulin A antibody to chlamydial major outer membrane protein antigen, chlamydial heat shock protein 60 and 10 antigens were measured by an enzyme-linked immunosorbent assay. In asymptomatic women, chlamydial load showed significant positive correlations with CD4, mDCs, interleukin-12 (IL-12) and IL-2; however, negative correlations were found with CD8 and IL-8 levels. In women with MPC, chlamydial IFUs correlated positively with CD8, pDC number, IL-8, CRP and interferon-gamma (IFN-gamma). In women with FD, chlamydial load showed a significant positive correlation with the pDC number, IL-10 and estradiol level and a negative correlation with CD4 and IFN-gamma. Overall, these results suggest that the interplay between chlamydial infectious load and host immune responses may be the deciding factor for the clinical condition presented during Chlamydia trachomatis infection.

The mobilization of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) to the cervix during chlamydial infection is not fully understood, and the role of these cells in immunopathogenesis is largely unknown. As an effective vaccine to control chlamydial infection is currently unavailable, understanding the regulation of the local immune response becomes a necessity. Therefore, mDC and pDC populations were analysed in peripheral blood and cervical samples of controls and Chlamydia-positive women, with or without mucopurulent cervicitis (MPC). Cervical cytokines and C-reactive protein levels in serum were quantified by ELISA and the chlamydial infectious load by culture. Chlamydia trachomatis infection mobilized both mDCs and pDCs to the cervical mucosa. pDCs were recruited more often in women with MPC (p <0.05) and they correlated significantly with the chlamydial load, C-reactive protein levels and cervical interleukin-8 (IL-8) levels. Upregulation of surface expression of co-stimulatory molecules (CD80, CD83 and CD86) on cervical mDCs and pDCs was observed during chlamydial infection but was significant only for mDCs. Significantly higher levels of IL-1 beta, IL-6 and IL-8 were observed in Chlamydia-positive women with MPC; however, after therapy, IL-8 levels decreased significantly. Median numbers of mDCs after therapy were significantly higher in the cervix and blood of infected women as compared to the numbers of pDCs, which were found to be lower in the cervix after therapy. These results thus suggest that during chlamydial infection, both mDCs and pDCs are recruited to the cervix, but their number and possible immunological functions may differ with the pathological condition. pDCs were associated more often with MPC and inflammatory factors, suggesting that they may possibly be involved in the immunopathogenesis of infections due to Chlamydia.

Background/aims: The objective of the present study was to examine the possible relationship between the chlamydial heat shock proteins (cHSP) 60 and 10 expression and the damaging sequelae of a Chlamydia trachomatis infection, such as infertility., Methods: Seven fertile and 7 infertile female patients infected with C. trachomatis attending the gynecology outpatient department of Safdarjung hospital (New Delhi, India) were enrolled. The relative transcript levels and intracellular expression of cHSP60 and cHSP10 in cervical cells were assessed using real-time RT-PCR and flow cytometry, respectively., Results: Quantitative real-time RT-PCR analysis showed that transcript levels of both cHSP60 (p = 0.007) and cHSP10 (p = 0.0006) were higher in infertile women than in fertile women. Flow cytometric analysis showed significantly higher intracellular levels of cHSP60 (p = 0.0006) and cHSP10 (p = 0.0041) in fertile women infected with Chlamydia than in infertile women. However, the percentage of double-positive cells (both cHSP60- and cHSP10-expressing cells) were higher (p = 0.0006) in infertile women than in fertile women., Conclusion: Our results suggest that cHSP60 and cHSP10 have a different pattern of expression in infertile women compared to fertile women reflecting a probable difference in the metabolic state of Chlamydia with the presence of an abnormal cryptic form of C. trachomatis in infertile women.

The role of major outer membrane protein (MOMP) variable regions in the interaction of chlamydiae and host cells has been evaluated and their role in neutralization of antibodies has been clearly demonstrated. There are also studies that delineate the contribution of these regions to the cell-mediated immune response of the host and suggest that serovar E elicits serovar-specific immune responses in infected humans. However, further studies with other serovars are required to confirm these findings and to elucidate the role and importance of serovar-specific responses of variable regions of MOMP in other serovars. We, therefore, performed a detailed analysis of the humoral and cellular immune responses against the serovar D-specific variable segments (VS) of MOMP in women infected with Chlamydia trachomatis. We found that VS4 elicits significantly higher responses (both humoral and cellular) than other VS peptides (VS1, VS2 and VS3). VS4 elicited significantly higher (P < 0.0001) proliferative responses, interferon-gamma levels (P < 0.0001) as well as higher prevalence (P < 0.0001) of IgG antibodies against VS4 in serovar D-infected patients as compared to patients infected with other serovars, suggesting its role in serovar-specific immune responses.

Background: Chlamydia trachomatis infection of the female genital tract can lead to serious sequelae resulting in fertility related disorders. Little is known about the mechanism leading to Chlamydia induced pathology and factors responsible for it. As only some of the women develops reproductive disorders while majority of the women clears infection without any severe sequalae, mucosal immune response in women with or without fertility disorders was studied to identify factors which may lead to final clinical outcome of chlamydial infection., Methods: Myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) populations in cervical mucosa and peripheral blood were analyzed in controls and Chlamydia positive women with or without fertility disorders with multicoloured flow cytometric analysis. Cervical cytokines (IL-6, IL-8, IL-10, IL-12, TNF-alpha and IFN-gamma), C-reactive protein levels and sex hormone levels in serum were quantified by ELISA., Results: In cervix of Chlamydia positive women with fertility disorders, significantly high (P < 0.05) numbers of pDCs were present with increased CD80 expression. pDCs correlated significantly with C-reactive protein levels, IL-6 and IFN-gamma levels in women with fertility disorders. In contrast, mDCs showed significant upregulation of CD1a during chlamydial infection and correlated significantly with IL-12 levels in Chlamydia positive fertile women. beta-estradiol levels were significantly higher in women having fertility disorders as compared to fertile women and have significant correlations (r = 0.65; P < 0.05) with pDCs numbers, CD80 expression, IL-6 levels and IFN-gamma levels in these women., Conclusion: These results suggest that development of sequalae in some women can be a result of interplay of many factors including type of dendritic cell, co stimulatory molecule expression, cytokine secretion pattern and hormone levels.

Background: The magnitude of reproductive morbidity associated with sexually transmitted Chlamydia trachomatis infection is enormous. A predictive serological test for upper genital tract infection would be a desirable diagnostic tool as C. trachomatis infection may lead to various immunopathological sequelae such as infertility, or ectopic pregnancy., Materials and Methods: Female patients (n = 198) attending gynecology outpatient department of Safdarjung hospital were enrolled for the study and clinically characterized into four groups on the basis of their symptoms; discharge, chronic cervicitis, primary and secondary infertility. Serological detection of C. trachomatis was done by ELISA using specific peptide sequences of major outer membrane protein (MOMP), Chlamydia heat shock protein (cHSP60 and 10)., Results: Significant high seropositivity to chlamydial anti-cHSP60 antibodies were detected in patients with secondary infertility. A significant percent of chlamydial reinfection was observed in patients having secondary infertility (82.6%; p < 0.01) and chronic cervicitis (64.28%; p < 0.05). Considering IgG MOMP ELISA as a test standard, anti-cHSP60 antibodies showed higher sensitivity (90.91%) and specificity (89.47%) than cHSP10 ELISA (75.6% and 73.87%) in the secondary infertile group. Further anti-cHSP60 antibodies' detection had a sensitivity of 67.33% and a specificity of 90.67% in secondary infertile women when compared with DFA and PCR., Conclusions: Our data suggest that detection of anti-cHSP60 antibodies would help in early prognosis of immunopathological sequelae in C. trachomatis-infected women and thereby in instituting appropriate therapy for controlling C. trachomatis infection at an early stage.

Background: The magnitude of reproductive morbidity associated with sexually transmitted Chlamydia trachomatis infection is enormous. Association of antibodies to chlamydial heat shock proteins (cHSP) 60 and 10 with various disease sequelae such as infertility or ectopic pregnancy has been reported. Cell-mediated immunity is essential in resolution and in protection to Chlamydia as well as is involved in the immunopathogenesis of chlamydial diseases. To date only peripheral cell mediated immune responses have been evaluated for cHSP60. These studies suggest cHSPs as important factors involved in immunopathological condition associated with infection. Hence study of specific cytokine responses of mononuclear cells from the infectious site to cHSP60 and cHSP10 may elucidate their actual role in the cause of immunopathogenesis and the disease outcome., Methods: Female patients (n = 368) attending the gynecology out patient department of Safdarjung hospital, New Delhi were enrolled for the study and were clinically characterized into two groups; chlamydia positive fertile women (n = 63) and chlamydia positive infertile women (n = 70). Uninfected healthy women with no infertility problem were enrolled as controls (n = 39). cHSP60 and cHSP10 specific cytokine responses (Interferon (IFN)-gamma, Interleukin (IL)-10, Tumor Necrosis Factor (TNF)-alpha, IL-13 and IL-4) were assessed by ELISA in stimulated cervical mononuclear cell supernatants., Results: cHSP60 and cHSP10 stimulation results in significant increase in IFN-gamma (P = 0.006 and P = 0.04 respectively) and IL-10 levels (P = 0.04) in infertile group as compared to fertile group. A significant cHSP60 specific increase in TNF-alpha levels (P = 0.0008) was observed in infertile group as compared to fertile group. cHSP60 and cHSP10 specific IFN-gamma and IL-10 levels were significantly correlated (P < 0.0001, r = 0.54 and P = 0.004, r = 0.33 respectively) in infertile group., Conclusion: Our results suggest that exposure to chlamydial heat shock proteins (cHSP60 and cHSP10) could significantly affect mucosal immune function by increasing the release of IFN-gamma, IL-10 and TNF-alpha by cervical mononuclear cells.

To increase the sensitivity and reliability of immunodiagnostic assay for direct detection of chlamydial genital infection, we developed monoclonal antibodies (MAbs) that recognize the major outer membrane protein (MOMP) of all serovars of Chlamydia trachomatis. ELISA and dot-ELISA were used to select hybridomas that produced anti-C. trachomatis MAbs. Four MAb clones--A12.6, B serogroup specific; C1.5, C serogroup specific; G10.2, species specific; and E9.1, genus specific--were characterized by SDS-PAGE and immunoblot and were reactive with MOMP of C. trachomatis. The species-specific MAb G10.2 had a molecular mass of 40 kDa. The reactivity of developed species-specific monoclonal antibody for detection of C. trachomatis antigen in endocervical specimens was compared with the commercially available DFA test. We found that developed antibody had high sensitivity (97.22%) compared to DFA for detection of chlamydial infection in endocervical samples. It can be used as a reliable method in laboratories for the diagnosis of chlamydial infections.

Most of the studies on 60-kDa and 10-kDa chlamydial heat shock proteins (HSPs) to date have been carried out with blood lymphocytes or serum antibody responses, which do not provide a clear picture of the actual pathogenesis as they do not differentiate primary infection from recurrent infection. Thus, in the present study induction of the immune response was evaluated by studying lymphoproliferation of both cervical and peripheral lymphocytes to synthetic peptides of cHSP60, cHSP10 and major outer membrane protein (MOMP) antigen. In addition, cervical antibody prevalence to MOMP antigen, cHSP60 and cHSP10 and cytokine levels in cervical washes was also determined. Positive proliferative responses of cervical lymphocytes to cHSP10 peptide were significantly higher (P < 0.05) in women with recurrent infections and that to MOMP antigen were significantly higher in primary infection. On proliferation of PBMCs with the above antigens, no significant difference was observed between primary and recurrent infection. Prevalence of cervical IgG and IgA antibodies to Chlamydia trachomatis was significantly higher (P < 0.05) during primary infection than recurrent infections. In contrast, prevalence of IgG and IgA antibodies to cHSP10 and IgG antibodies to cHSP60 was higher during recurrent infections than primary infections. Interferon (IFN)-gamma levels were significantly higher in cervical washes of women with recurrent infection and correlated strongly with cHSP60 antibody titres. Our data thus suggest that mucosal responses are more appropriate in understanding the pathogenesis of chlamydial infection and IFN-gamma could be involved in the modulation of immune responses towards chlamydial infection directly, by causing acute inflammation, or indirectly through modulation of HSP expression.

Chlamydia trachomatis infection is followed by the development of antigen-specific cell-mediated immunity, which is detectable as a positive lymphocyte proliferation response to the chlamydial major outer membrane protein (MOMP) antigen. To date, however, there have been no studies on the mucosal immune responses to chlamydial antigens. This study aimed to study the primary and secondary immune responses of cervical lymphocytes in response to the chlamydial antigen. Median proliferative responses were found to be significantly (P<0.05) higher in patients with chlamydial infections than in controls. The chlamydial MOMP induced significantly higher IL-6 and IL-10 and lower interferon-gamma (IFN-gamma) secretion in cervical lymphocytes of Chlamydia-positive women, resulting in a T helper 2 response. On stimulation of peripheral blood mononuclear cells (PBMC) obtained from Chlamydia-positive women with the chlamydial antigen, the median levels of IL-10, IL-12 and IFN-gamma were higher than in controls, but the differences were not significant. Our study suggests that the mucosal immune responses towards Chlamydia trachomatis are different from those of PBMCs and are more helpful in understanding the cytokine responses in the female genital tract during chlamydial infection.

Little is known about concurrent expression of cervical cytokines and their regulation by sex hormones during primary or recurrent chlamydial infections in humans. Cytokine (interleukin-1beta [IL-1beta], IL-6, IL-10, interferon-gamma [IFN-gamma], and tumor necrosis factor-alpha [TNF-alpha]) concentrations in cervical washes and serum samples, along with levels of beta-estradiol and progesterone in women with primary or recurrent chlamydial infections and healthy controls, were measured by ELISA. Women with recurrent infections had significantly higher levels of IFN-gamma in cervical washes than did women with primary infections. Significant negative correlation was found between IL-1beta and progesterone levels during recurrent infections. Beta-estradiol levels in women with primary infections showed significant negative correlations with cervical concentrations of IL-10, IL-1beta, and IL-6. Our study suggests that Chlamydia trachomatis infection in the female genital tract may be regulated by both the synergistic actions of the cytokines and the sex hormones beta-estradiol and progesterone.

Problem: Chlamydial infections are often associated with various fertility-related disorders. Serological prediction of these has limitations, as they do not differentiate between past and current infections. Thus, we looked for local markers that could predict more precisely women at higher risk of developing severe complications., Method of Study: A total of 320 Chlamydia trachomatis positive women with or without fertility disorders were tested for the prevalence of immunoglobulin A antibodies to synthetic peptides of chlamydial heat-shock protein 60 (cHSP60) and cHSP10 along with cervical interferon-gamma (IFN-gamma) and serum C-reactive protein (CRP) levels., Results: Positive IFN-gamma level was the single best predictor for fertility disorder [odds ratio (OR) 15.4]. The predictive value of IFN-gamma could be significantly improved only by the addition of CRP test (OR 37.9)., Conclusion: Positive IFN-gamma levels in cervical washes along with elevated CRP levels could be used to predict women who are at higher risk of developing fertility disorders.

Persistent, untreated chlamydial infection causes chronic stimulation of the host immune system against immunogenic antigens such as chlamydial heat shock proteins (cHSP) 60 and 10. In order to find the seroprevalence of antibodies to cHSPs, enzyme-linked immunosorbent assay (ELISA) is performed using specific peptide sequences to measure antibody response against major outer membrane protein (MOMP), cHSP60 and cHSP10 in patient sera. In this study, 255 patients attending the gynaecology out-patient department (March 2004 to August 2005) of Safdarjung Hospital were enrolled. Of these patients, 107 were diagnosed with cervicitis while 52 had pelvic inflammatory disease (PID)/infertility. Chlamydia trachomatis infection in endocervical specimens is diagnosed by a direct fluorescence assay (DFA) and the polymerase chain reaction (PCR). In 75 (29.4%) of the C. trachomatis-positive women, 50 (66.7%) were ELISA positive for MOMP 48 (64.0%) were positive for cHSP60 and 46 (61.3%) were positive for cHSP10. The anti-MOMP index correlated positively with anti-cHSP60 (R = 0.522, P < 0.01) and anti-cHSP10 (R = 0.286, P < 0.05). Antibody titre for MOMP was significantly higher than that for cHSP60 (1:5; P < 0.01 and 1:25; P < 0.05). Moreover, patients with PID/infertility showed significantly higher antibody titres for cHSP60 and cHSP10 when compared to patients with cervicitis at dilutions of 1 in 50, 1 in 250, 1 in 1250 (P < 0.001) and at 1 in 6250 (P < 0.01).

Objective: To use fluorescence in situ hybridization (FISH) using ribosomal RNA (rRNA) oligonucleotide probes as the target nucleic acid for the detection of Chlamydia trachomatis., Study Design: Suitable sequences selected from the rRNA sequence of C trachomatis were labeled with a fluorescent dye and used in FISH for detecting chlamydial inclusion bodies and/ or elementary bodies in paraformaldehyde-fixed urogenital swab samples. The sensitivity and specificity of the FISH assay were compared with those of the polymerase chain reaction (PCR) using plasmid primers. Positive known C trachomatis-infected McCoy cells were used as positive controls. Urogenital swab specimens that were C trachomatis negative on culture and PCR were used as negative controls., Result: Among the 128 samples included in the study, FISH was positive in 28 (21.8%) and PCR in 33 (25.7%). A significant correlation was found between the 2 detection methods. Results of PCR and FISH were consistent in 115 of the 128 samples (R = 0.89). Thirteen samples showed discordant results. Of these, 9 FISH negative samples were PCR positive and 4 FISH positive samples were PCR negative., Conclusion: FISH was a highly specific and fairly sensitive technique for detecting C trachomatis. Signal amplification techniques and use of different fluorophores may further increase the sensitivity of this technique.

Human genital infection caused by Chlamydia trachomatis is thought to be immunologically mediated, resulting in local recruitment of lymphocyte subsets and inducing the production of cytokines. Little information is available about the role of lymphocyte recruitment and the regulation of cytokine production in the genital tract of C. trachomatis positive infertile women. We have evaluated the recruitment of lymphocyte subsets in the genital tract and production of Th1/Th2 cytokines in cervical secretions and laparoscopic specimens from the fallopian tubes of C. trachomatis positive infertile women (n = 17) and compared them with controls, viz. C. trachomatis negative infertile women (n = 20) using ELISA and flow cytometry. None of these patients were found to be infected either with Candida sps., bacterial vaginosis, Trichomonas vaginalis, Neisseria gonorrhoeae, Mycoplasma hominis or Ureaplasma urealyticum in the cervix. Flow cytometric analysis of cervical secretions in Chlamydia positive women revealed recruitment of both CD4 and CD8 lymphocytes to the genital tract was up-regulated and a variation in the production rates of different cytokines in cervical secretions and fallopian tube was observed. We found that the immune responses in cervical secretions were of Th0 type, since all the analysed cytokines, viz. IFN-gamma, TNF-alpha, IL-10 and IL-12 were up-regulated. As, both CD4 and CD8 cells contribute to the production of IFN-gamma and IL-10, these results suggest that along with CD4 cells, CD8 lymphocytes also may be important for local regulation of Th1/Th2 responses in the genital tract during C. trachomatis infection.

Objective: To investigate changes in cell-mediated immunophenotypes by flow cytometry in endocervical secretions and peripheral blood in women with Chlamydia trachomatis infection., Study Design: Fifty women attending the gynaecology outpatient department of Safdarjang Hospital, New Delhi, India, with signs and symptoms of cervicitis were enrolled. All patients underwent endocervical screening for C trachomatis (direct fluorescence antibody test [DFA]), and any coinfection with Candida (Gram stain), bacterial vaginosis (Gram stain), Neisseria gonorrhoeae (Gram stain), Trichomonas vaginalis (wet mount) and HIV (enzyme-linked immunosorbent assay) was ruled out. Flow cytometry was done to investigate changes in immunophenotypes in endocervical secretions and peripheral blood using monoclonal antibodies for surface markers (CD3, CD4, CD8, CD19, CD45 and CD83). Data were analyzed by chi2 test, while means were compared using Student's t test., Results: C trachomatis positivity was found to be 36% (n = 18). Forty-eight patients constituted the study population since 2 patients coinfected with Candida, bacterial vaginosis and T vaginalis were excluded. A statistically significant enhancement in CD4+, CD8+ and dendritic cellular phenotypes was observed in the endocervical secretions of Chlamydia-positive patients, while B cells showed no marked difference. In the parallel study of matched peripheral blood, immunophenotypes did not show statistically significant results., Conclusion: Increased influx of CD4+, CD8+ and dendritic cells in the endocervix is an indication of cell-mediated immunity in response to C trachomatis infection. Local immune response in the cervical region is independent of systemic response. The mechanism by which local mucosal and systemic immune cells interact to repel or enhance susceptibility to C trachomatis infection requires further study.

Screening for Chlamydia trachomatis was done for 280 endocervical swab samples by PCR specific for endogenous plasmid. Age dependency was seen in symptomatic patients, with a high chlamydial prevalence rate (28%) found in younger women. Genotyping by restriction fragment length polymorphism analysis of omp1 PCR-positive samples showed serovars D, E, and F to be the most prevalent.

Objectives: To screen and treat chlamydial infection in pregnant women in order to assess the effects of therapeutic intervention on the outcome of Chlamydia trachomatis-infected pregnancy., Methods: Enrolled in the study were 350 women in the first to third trimesters of pregnancy. Endocervical swabs were collected for C. trachomatis diagnosis by DFA and PCR. A few STD infections, viz.: Neisseria gonorrhoeae, Candida spp., bacterial vaginosis, Trichomonas vaginalis and Treponema pallidum were ruled out. After excluding 22 patients infected with other sexually transmitted infections, a cohort of 328 pregnant women comprised the study population. Anti-chlamydial treatment (viz.: oral therapy with erythromycin stearate, 500 mg 4 times daily for 7 days) was given to 17 women (group I) and their partners. Fifteen patients of group I were retested by DFA and PCR assay for C. trachomatis infection 2 weeks following therapy. Other Chlamydia-positive patients (n=42) who were lost to follow-up were classified as untreated positive cases (group II) while group III included C. trachomatis negative cases (n=269). Data on obstetric outcome were recorded in a total of 164 cases. Statistical comparison of the data were done using the chi(2)-test and means were compared using Student's t-test., Results: Among the 350 pregnant women enrolled initially for the study, C. trachomatis positivity was found to be 18.8% (n=66) in the endocervix by DFA and PCR assay. Co-infection with Candida spp., bacterial vaginosis, T. vaginalis and T. pallidum was 2.0%, 1.7%, 1.7% and 0.5%, respectively. None of the pregnant women was infected with N. gonorrhoeae. Pregnant women at an increased risk of chlamydial infection included those who had multiple births and were in second trimester of pregnancy. Fifteen patients of group I became Chlamydia-negative following treatment. Data on obstetric outcome were recorded in 11, 26 and 127 patients of groups I, II and III, respectively. The mean duration of gestation for premature deliveries was found to be significantly higher in group I in comparison with group II [35.5 vs. 33.1 weeks (P<0.05)], thereby showing an improved effect of treatment on pregnancy outcome. The mean of low birth weight births was higher in group I compared with group II (2200.0 vs. 2113.3 g), however, this was statistically non-significant. Stillbirths were significantly higher among group II in comparison with group III [11.5% vs. 4.7% (chi(2)=1.79; P<0.5)]. No stillbirths were recorded in patients who had taken anti-chlamydial treatment., Conclusions: Our findings suggest that routine screening and treatment of C. trachomatis infection in pregnant women, especially those in high risk groups, should be mandatory to reduce the adverse effects on obstetric outcome.

Objectives: To detect Chlamydia trachomatis infection by polymerase chain reaction (PCR) in symptomatic women attending a gynecology clinic in a city hospital and in randomly selected slum dwellers., Study Design: Endocervical specimens were collected from 350 women with genitourinary complaints (group I) and 53 slum dwellers (group II). Samples were analyzed by PCR, direct fluorescence assay (DFA) and Giemsa stain cytology for detection of C trachomatis and compared for their sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV)., Results: The prevalence of endocervical C trachomatis infection was 43.1% and 24.5% in groups I and II, respectively. The sensitivity, specificity, PPV and NPV of PCR were 80.0%, 75.0%, 66.6% and 85.7%, respectively, when DFA was considered true positive. The percent increment in detection of C trachomatis by PCR was 15.3%., Conclusion: Giemsa stain cytology has low sensitivity and specificity; hence, it cannot be recommended for use as a diagnostic technique. It appears that PCR can be used routinely in Chlamydia diagnosis and in screening selected populations. The high positivity of C trachomatis infection in urban slum dwellers is cause for concern.

In order to investigate whether or not Chlamydia trachomatis infection is a risk factor for pregnancy loss, 77 spontaneous abortion patients (6-24 weeks gestation), admitted to gynaecology emergency of Safdarjang Hospital, New Delhi, India. Twenty-five pregnant women (6-16 weeks gestation) attending the same hospital for induced abortion, were included in the study. C. trachomatis antigen was detected in endometrial curretage tissue by enzyme immunoassay (EIA). The detection rate was 15.6% (12/77) among spontaneous abortion patients and 4% (1/25) among women undergoing induced abortion. There was no statistically significant association between the mean age/mean gestational age of those experiencing spontaneous abortion, with and without C. trachomatis infection (26.9 years versus 25.06 years and 11.1 weeks versus 9.6 weeks, respectively). High prevalence of C. trachomatis was found in multigravidae and parous spontaneous abortion patients, compared with that in primigravidae and nulliparous Chlamydia-negative spontaneous aborters (75.0% versus 25.0%; 66.7% versus 33.3%, respectively). The prevalence of chlamydial antigen in patients with no prior history of spontaneous abortion was 16.1% (10/62) compared with 18.1% (2/11) in women with one prior abortion. Further study is required to determine whether C. trachomatis infection is a primary or secondary indicator of risk.

A cohort of 122 pregnant women attending the hospital antenatal clinic in northern India were studied to determine the prevalence of genital chlamydial infection, and any adverse effect on the pregnancy. Endocervical swabs were taken at > 12 weeks of pregnancy and cultured for Chlamydia trachomatis. Twenty-six (21.3%) pregnant women were found to be infected with C. trachomatis. The mean age, gravidity and parity were significantly higher (25.03 vs 23.6 years, 1.88 vs 1.72 and 0.92 vs 0.68 respectively [P < 0.005]) in women from whom C. trachomatis was isolated. Follow-up was possible in 87 women who delivered in the hospital. There was increased incidence of still-birth, prematurity and low birth-weight in the C. trachomatis-positive women (16.6% vs 5.7%, 26.6% vs 18.4% and 26.6% vs 23.0%), and these differences were statistically significant (P < 0.5, P < 0.5 and P < 0.05 respectively). The results suggest a definite need for C. trachomatis screening on a wider scale, both in different risk groups of asymptomatic antenatal women and in neonates, to confirm these findings.