Aruna Mittal

Cytokine expression pattern in the genital tract of Chlamydia trachomatis positive infertile women – implication for T-cell responses

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‐γ, TNF‐α, IL‐10 and IL‐12 were up‐regulated. As, both CD4 and CD8 cells contribute to the production of IFN‐γ 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.

Decreased susceptibility to azithromycin and doxycycline in clinical isolates of Chlamydia trachomatis obtained from recurrently infected female patients in India.

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.

Predominance of Chlamydia trachomatis Serovars Associated with Urogenital Infections in Females in New Delhi, India

ABSTRACT 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.

Effect of treatment for Chlamydia trachomatis during pregnancy

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 χ2‐test and means were compared using Students 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% (χ2=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.

Mucosal and peripheral immune responses to chlamydial heat shock proteins in women infected with Chlamydia trachomatis

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)‐γ 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‐γ could be involved in the modulation of immune responses towards chlamydial infection directly, by causing acute inflammation, or indirectly through modulation of HSP expression.

Pattern of Interleukins in Minimal-Change Nephrotic Syndrome of Childhood

Assays of interleukin-1 (IL-1) and IL-2 were done in supernatants from phytohaemagglutinin-activated lymphocyte cultures from 10 children suffering from minimal-change nephrotic syndrome (MCNS) to assess their role in the aetiopathogenesis of this disorder. Increased levels of IL-1 and IL-2 had been found in supernatants from patients having MCNS compared with controls, suggesting a significant role of these cytokines in the immunopathogenesis of proteinuria in this syndrome.

High immunoglobulin A seropositivity for combined Chlamydia pneumoniae, Helicobacter pylori infection, and high-sensitivity C-reactive protein in coronary artery disease patients in India can serve as atherosclerotic marker

Atherosclerosis is increasingly recognized as a chronic inflammatory disease. A variety of infectious agents (Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus [CMV]) and inflammatory marker such as high-sensitivity C-reactive protein (hs-CRP) have been found to be associated with atherosclerosis and its consequences. There is a need to know about the type and burden of infection in coronary artery disease (CAD) patients and the level of hs-CRP in India as there is growing evidence that a variety of pathogens are participating in the development and/or acceleration of at least pre-existing atherosclerosis. In addition, there is a need to find the association between these pathogens and conventional risk factors among CAD patients in India, to possibly identify a prognostic marker. In this study 192 patients with incident or prevalent CAD attending the Cardiology Outpatient Department of Safdarjung Hospital, New Delhi, India, were enrolled. In addition, 192 age-and sex-matched controls were also included. Cases and controls differ significantly in seropositivity to C. pneumoniae immunoglobulin IgA (154 vs 76) and IgG (71 vs 48) (P < 0.001, P < 0.015), H. pylori IgA (98 vs 57) and IgG (77 vs 43) (P < 0.001, P < 0.001), CMV IgG (62 vs 38) (P = 0.01) and with hs-CRP (114 vs 60) (P < 0.001), respectively. The level of hs-CRP was higher in CAD patients with IgA seropositivity of C. pneumoniae and H. pylori (5.18 and.65 mg/l) than the IgG of these bacteria (3.73 and 3.36 mg/l), respectively. These findings support an association between specific infectious agents, namely, C. pneumoniae, H. pylori, CMV, and hs-CRP in CAD patients. Association of hs-CRP with IgA specific for C. pneumoniae and H. pylori suggests the role of chronic infection in the development of CAD and may be used as a marker to target the population.

In infertile women, cells from Chlamydia trachomatis infected site release higher levels of interferon-gamma, interleukin-10 and tumor necrosis factor-alpha upon heat shock protein stimulation than fertile women

BackgroundThe 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.MethodsFemale 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.ResultscHSP60 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.ConclusionOur 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.

PROTECTIVE OR PATHOGENIC IMMUNE RESPONSE TO GENITAL CHLAMYDIAL INFECTION IN WOMEN-A POSSIBLE ROLE OF CYTOKINE SECRETION PROFILE OF CERVICAL MUCOSAL CELLS

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.

Polymerase Chain Reaction for Detection of Endocervical Chlamydia trachomatis Infection in Women Attending a Gynecology Outpatient Department in India

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.