Shift work influences the outcomes of Chlamydia infection and pathogenesis.

Shift work, performed by approximately 21 million Americans, is irregular or unusual work schedule hours occurring after 6:00 pm. Shift work has been shown to disrupt circadian rhythms and is associated with several adverse health outcomes and chronic diseases such as cancer, gastrointestinal and psychiatric diseases and disorders. It is unclear if shift work influences the complications associated with certain infectious agents, such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility resulting from genital chlamydial infection. We used an Environmental circadian disruption (ECD) model mimicking circadian disruption occurring during shift work, where mice had a 6-h advance in the normal light/dark cycle (LD) every week for a month. Control group mice were housed under normal 12/12 LD cycle. Our hypothesis was that compared to controls, mice that had their circadian rhythms disrupted in this ECD model will have a higher Chlamydia load, more pathology and decreased fertility rate following Chlamydia infection. Results showed that, compared to controls, mice that had their circadian rhythms disrupted (ECD) had higher Chlamydia loads, more tissue alterations or lesions, and lower fertility rate associated with chlamydial infection. Also, infected ECD mice elicited higher proinflammatory cytokines compared to mice under normal 12/12 LD cycle. These results imply that there might be an association between shift work and the increased likelihood of developing more severe disease from Chlamydia infection.

Epidermal Growth Factor Receptor and Transforming Growth Factor ß Signaling Pathways Cooperate To Mediate Chlamydia Pathogenesis.

Human genital Chlamydia infection is a major public health concern due to the serious reproductive system complications. Chlamydia binds several receptor tyrosine kinases (RTKs) on host cells, including the epidermal growth factor receptor (EGFR), and activates cellular signaling cascades for host invasion, cytoskeletal remodeling, optimal inclusion development, and induction of pathogenic epithelial-mesenchyme transition (EMT). Chlamydia also upregulates transforming growth factor beta (TGF-ß) expression, whose signaling pathway synergizes with the EGFR cascade, but its role in infectivity, inclusions, and EMT induction is unknown. We hypothesized that the EGFR and TGF-ß signaling pathways cooperate during chlamydial infection for optimal inclusion development and stable EMT induction. The results revealed that Chlamydia upregulated TGF-ß expression as early as 6 h postinfection of epithelial cells and stimulated both the EGFR and TGF-ß signaling pathways. Inhibition of either the EGFR or TGF-ßR1 signaling substantially reduced inclusion development; however, the combined inhibition of both EGFR and TGF-ßR1 signaling reduced inclusions by over 90% and prevented EMT induction. Importantly, EGFR inhibition suppressed TGF-ß expression, and an inhibitory thrombospondin-1 (Tsp1)-based peptide inhibited chlamydia-induced EMT, revealing a major source of active TGF-ß during infection. Finally, TGF-ßR signaling inhibition suppressed the expression of transforming acidic coiled-coil protein-3 (TACC3), which stabilizes EGFR signaling, suggesting reciprocal regulation between TGF-ß and EGFR signaling during chlamydial infection. Thus, RTK-mediated host invasion by chlamydia upregulated TGF-ß expression and signaling, which cooperated with other cellular signaling cascades and cytoskeletal remodeling to support optimal inclusion development and EMT induction. This finding may provide new targets for chlamydial disease biomarkers and prevention.

A unique insight into the MiRNA profile during genital chlamydial infection.

BACKGROUND: Genital C. trachomatis infection may cause pelvic inflammatory disease (PID) that can lead to tubal factor infertility (TFI). Understanding the pathogenesis of chlamydial complications including the pathophysiological processes within the female host genital tract is important in preventing adverse pathology. MicroRNAs regulate several pathophysiological processes of infectious and non-infectious etiologies. In this study, we tested the hypothesis that the miRNA profile of single and repeat genital chlamydial infections will be different and that these differences will be time dependent. Thus, we analyzed and compared differentially expressed mice genital tract miRNAs after single and repeat chlamydia infections using a C. muridarum mouse model. Mice were sacrificed and their genital tract tissues were collected at 1, 2, 4, and 8 weeks after a single and repeat chlamydia infections. Histopathology, and miRNA sequencing were performed. RESULTS: Histopathology presentation showed that the oviduct and uterus of reinfected mice were more inflamed, distended and dilated compared to mice infected once. The miRNAs expression profile was different in the reproductive tissues after a reinfection, with a greater number of miRNAs expressed after reinfection. Also, the number of miRNAs expressed each week after chlamydia infection and reinfection varied, with weeks eight and one having the highest number of differentially expressed miRNAs for chlamydia infection and reinfection respectively. Ten miRNAs; mmu-miR-378b, mmu-miR-204-5p, mmu-miR-151-5p, mmu-miR-142-3p, mmu-miR-128-3p, mmu-miR-335-3p, mmu-miR-195a-3p, mmu-miR-142-5p, mmu-miR-106a-5p and mmu-miR-92a-3p were common in both primary chlamydia infection and reinfection. Pathway analysis showed that, amongst other functions, the differentially regulated miRNAs control pathways involved in cellular and tissue development, disease conditions and toxicity. CONCLUSIONS: This study provides insights into the changes in miRNA expression over time after chlamydia infection and reinfection, as well as the pathways they regulate to determine pathological outcomes. The miRNAs networks generated in our study shows that there are differences in the focus molecules involved in significant biological functions in chlamydia infection and reinfection, implying that chlamydial pathogenesis occurs differently for each type of infection and that this could be important when determining treatments regime and disease outcome. The study underscores the crucial role of host factors in chlamydia pathogenesis.

The immunoregulatory role of alpha enolase in dendritic cell function during Chlamydia infection.

BACKGROUND: We have previously reported that interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells that induced elevated protective immunity against Chlamydia. To further investigate the molecular and biochemical mechanism underlying the superior immunostimulatory property of IL-10 deficient DCs we performed proteomic analysis on protein profiles from Chlamydia-pulsed wild-type (WT) and IL-10-/- DCs to identify differentially expressed proteins with immunomodulatory properties. RESULTS: The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the last step of glycolysis was significantly upregulated in Chlamydia-pulsed IL-10-/- DCs compared to WT DCs. We further studied the immunoregulatory role of ENO1 in DC function by generating ENO1 knockdown DCs, using lentiviral siRNA technology. We analyzed the effect of the ENO1 knockdown on DC functions after pulsing with Chlamydia. Pyruvate assay, transmission electron microscopy, flow cytometry, confocal microscopy, cytokine, T-cell activation and adoptive transfer assays were also used to study DC function. The results showed that ENO1 knockdown DCs had impaired maturation and activation, with significant decrease in intracellular pyruvate concentration as compared with the Chlamydia-pulsed WT DCs. Adoptive transfer of Chlamydia-pulsed ENO1 knockdown DCs were poorly immunogenic in vitro and in vivo, especially the ability to induce protective immunity against genital chlamydia infection. The marked remodeling of the mitochondrial morphology of Chlamydia-pulsed ENO1 knockdown DCs compared to the Chlamydia-pulsed WT DCs was associated with the dysregulation of translocase of the outer membrane (TOM) 20 and adenine nucleotide translocator (ANT) 1/2/3/4 that regulate mitochondrial permeability. The results suggest that an enhanced glycolysis is required for efficient antigen processing and presentation by DCs to induce a robust immune response. CONCLUSIONS: The upregulation of ENO1 contributes to the superior immunostimulatory function of IL-10 deficient DCs. Our studies indicated that ENO1 deficiency causes the reduced production of pyruvate, which then contributes to a dysfunction in mitochondrial homeostasis that may affect DC survival, maturation and antigen presenting properties. Modulation of ENO1 thus provides a potentially effective strategy to boost DC function and promote immunity against infectious and non-infectious diseases.

The Roles of Unfolded Protein Response Pathways in Chlamydia Pathogenesis.

Chlamydia is an obligate intracellular bacterium that relies on host cells for essential nutrients and adenosine triphosphate (ATP) for a productive infection. Although the unfolded protein response (UPR) plays a major role in certain microbial infectivity, its role in chlamydial pathogenesis is unknown. We hypothesized that Chlamydia induces UPR and exploits it to upregulate host cell uptake and metabolism of glucose, production of ATP, phospholipids, and other molecules required for its replicative development and host survival. Using a combination of biochemical and pathway inhibition assays, we showed that the 3 UPR pathway transducers-protein kinase RNA-activated (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1α (IRE1α), and activating transcription factor-6α (ATF6α)-were activated during Chlamydia infection. The kinase activity of PERK and ribonuclease (RNase) of IRE1α mediated the upregulation of hexokinase II and production of ATP via substrate-level phosphorylation. In addition, the activation of PERK and IRE1α promoted autophagy formation and apoptosis resistance for host survival. Moreover, the activation of IRE1α resulted in the generation of spliced X-box binding protein 1 (sXBP1) and upregulation of lipid production. The vital role of UPR pathways in Chlamydia development and pathogenesis could lead to the identification of potential molecular targets for therapeutics against Chlamydia.

Role of Epithelial-Mesenchyme Transition in Chlamydia Pathogenesis.

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy.

The use of biomarkers of semen exposure in sexual and reproductive health studies.

Biomarkers of semen exposure have been used in studies investigating the safety and efficacy of barrier methods of contraception. They have been used as objective indicators of semen exposure when studying sexual behaviors and in human immunodeficiency virus/sexually transmitted infection research interventions where participants are advised to avoid unprotected sex. Semen biomarkers have also been used to assess or validate self-reported sexual behaviors or condom use in reproductive health settings. Prostate-specific antigen (PSA) and Y chromosome DNA (Yc-DNA) have each been evaluated in the past as semen biomarkers and are the most widely used in the field. While both are considered reliable for evaluating exposure to semen, each has unique characteristics. In this report, we summarize the literature and provide some considerations for reproductive health researchers who are interested in using PSA or Yc-DNA as semen biomarkers. We also synthesize our previous published work on the optimal conditions of collecting and storing specimens and assay performance in the presence of other vaginal products that may influence various assays. Semen biomarkers are innovative and promising tools to further study and better understand women's reproductive and sexual health and behavior. More research is needed to better understand the strengths, limitations, and optimal performance conditions of specific assays in vivo.

Does tenofovir gel or do other microbicide products affect detection of biomarkers of semen exposure in vitro?

OBJECTIVES: There is currently no information on whether products evaluated in HIV microbicide trials affect the detection of the semen biomarkers prostate-specific antigen (PSA) or Y chromosome DNA. STUDY DESIGN: We tested (in vitro) dilutions of tenofovir (TFV), UC781 and the hydroxyethylcellulose (HEC) placebo gels using the Abacus ABAcard and the quantitative (Abbott Architect total PSA) assays for PSA and Y chromosome DNA by real-time polymerase chain reaction. RESULTS: TFV gel and the HEC placebo adversely affected PSA detection using the ABAcard but not the Abbott Architect total PSA assay. UC781 adversely affected both the ABAcard and Abbott Architect total PSA assays. While there were some quantitative changes in the magnitude of the signal, none of the products affected positivity of the Y chromosome assay. CONCLUSIONS: The presence of TFV or HEC gels did not affect quantitative PSA or Y chromosome detection in vitro. Confirmation of these findings is recommended using specimens obtained following use of these gels in vivo. IMPLICATIONS: Researchers should consider the potential for specific microbicides or any products to affect the particular assay used for semen biomarker detection. The ABAcard assay for PSA detection should not be used with TFV UC781, or HEC.

Effect of lubricants and a vaginal spermicide gel on the detection of prostate specific antigen, a biomarker of semen exposure, using a quantitative (Abbott ARCHITECT) assay.

OBJECTIVES: Little is known about the effects of commonly used lubricants on detection of biomarkers of semen exposure. We investigated the in vitro effect of Gynol®, K-Y Jelly®, Replens®, Astroglide®, Carbopol, and Silicorel on quantitative detection of prostate specific antigen (PSA). STUDY DESIGN: A predetermined concentration of each of the gels was added to serially diluted semen samples. Additionally, serial dilutions of each of the gels were added to three different semen dilutions (high, medium, or low). The resulting samples were tested for PSA on the Abbott ARCHITECT System. RESULTS: When using the Abbott ARCHITECT system, the only products that inhibited PSA detection were Gynol® and Replens®. The inhibition caused by Gynol® was dose-dependent, but that of Replens was dose-independent. K-Y Jelly®-spiked samples had higher PSA values than controls. CONCLUSIONS: Caution is warranted when using the Abbott quantitative assay for PSA detection as a biomarker of semen exposure in settings where Gynol®, Replens® or K-Y Jelly® might also have been used. Neither Astroglide® nor Silicorel inhibited PSA detection. Additional studies evaluating other vaginal products, including microbicides, and their effects on other assays, are needed. In vivo studies will be especially important to optimize PSA detection from clinical samples. IMPLICATIONS: Researchers should consider the potential for specific lubricants or any vaginal products to affect the particular assay used for semen biomarker detection. The Abbott ARCHITECT's total PSA assay should not be used with the product Replens. Caution is warranted when using the assay in settings where Gynol or K-Y jelly may have been used.

Detection of two biological markers of intercourse: prostate-specific antigen and Y-chromosomal DNA.

BACKGROUND: Although biological markers of women's exposure to semen from vaginal intercourse have been developed as surrogates for risk of infection or probability of pregnancy, data on their persistence time and clearance are limited. STUDY DESIGN: During 2006-2008, 52 couples were enrolled for three 14-day cycles of abstinence from vaginal sex during which women were exposed in the clinic to a specific quantity (10, 100 or 1000 µL) of their partner's semen. Vaginal swabs were collected before and at 1, 6, 12, 24, 48, 72 and 144 h after exposure for testing for prostate-specific antigen (PSA) and Y-chromosome DNA (Yc DNA). RESULTS: Immediately after exposure to 1000 µL of semen, the predicted sensitivity of being PSA positive was 0.96; this decreased to 0.65, 0.44, 0.21 and 0.07 at 6, 12, 24 and 48 h, respectively. Corresponding predicted sensitivity of being Yc DNA positive was 0.72 immediately postexposure; this increased to 0.76 at 1 h postexposure and then decreased to 0.60 (at 6 h), 0.63 (at 12 h), 0.49 (at 24 h), 0.21 (at 48 h), 0.17 (at 72 h) and 0.12 (at 144 h). CONCLUSIONS: Overall findings suggest that PSA may be more consistent as a marker of very recent exposure and that Yc DNA is more likely to be detected in the vagina after 12 h postexposure compared to PSA.

Prevention of Chlamydia-induced infertility by inhibition of local caspase activity.

Tubal factor infertility (TFI) represents 36% of female infertility and genital infection by Chlamydia trachomatis (C. trachomatis) is a major cause. Although TFI is associated with host inflammatory responses to bacterial components, the molecular pathogenesis of Chlamydia-induced infertility remains poorly understood. We investigated the hypothesis that activation of specific cysteine proteases, the caspases, during C. trachomatis genital infection causes the disruption of key fertility-promoting molecules required for embryo development and implantation. We analyzed the effect of caspase inhibition on infertility and the integrity of Dicer, a caspase-sensitive, fertility-promoting ribonuclease III enzyme, and key micro-RNAs in the reproductive system. Genital infection with the inflammation- and caspase-inducing, wild-type C. trachomatis serovar L2 led to infertility, but the noninflammation-inducing, plasmid-free strain did not. We confirmed that caspase-mediated apoptotic tissue destruction may contribute to chlamydial pathogenesis. Caspase-1 or -3 deficiency, or local administration of the pan caspase inhibitor, Z-VAD-FMK into normal mice protected against Chlamydia-induced infertility. Finally, the oviducts of infected infertile mice showed evidence of caspase-mediated cleavage inactivation of Dicer and alteration in critical miRNAs that regulate growth, differentiation, and development, including mir-21. These results provide new insight into the molecular pathogenesis of TFI with significant implications for new strategies for treatment and prevention of chlamydial complications.

Optimal methods for collecting and storing vaginal specimens for prostate-specific antigen testing in research studies.

BACKGROUND: Prostate-specific antigen (PSA) detected in vaginal fluid can be used in studies of HIV/sexually transmitted infection (STI) and pregnancy prevention as an alternative to relying on participant reports of exposure to semen. Optimal methods for collecting and storing specimens for this testing have not been determined. STUDY DESIGN: We conducted a controlled, in vitro experiment of 550 specimens spiked with semen to determine the effects of swab type (five types), storage conditions of the swabs (room temperature with or without desiccant or at -80°C without desiccant) and time from collection to testing (seven intervals over the course of 12 months) on the identification of PSA. We performed factorial analysis of variance to identify factors influencing PSA detection. RESULTS: Concentrations of PSA detected in the swabs declined with time of storage over the 1-year experiment (p<.01). The 1-mL, rayon-tipped swab stored immediately at -80°C following collection performed best. CONCLUSIONS: If immediate testing or freezer storage is not feasible, investigators should use a swab with 1-mL capacity with processing and testing as soon as possible after specimen collection.

Chlamydial infection in vitamin D receptor knockout mice is more intense and prolonged than in wild-type mice.

Vitamin D hormone (1,25-dihydroxyvitamin D) is involved in innate immunity and induces host defense peptides in epithelial cells, suggesting its involvement in mucosal defense against infections. Chlamydia trachomatis is a major cause of bacterial sexually transmitted disease worldwide. We tested the hypothesis that the vitamin D endocrine system would attenuate chlamydial infection. Vitamin D receptor knock-out mice (VDR(-/-)) and wild-type mice (VDR(+/+)) were infected with 10(3) inclusion forming units of Chlamydia muridarum and cervical epithelial cells (HeLa cells) were infected with C. muridarum at multiplicity of infection 5:1 in the presence and absence of 1,25-dihydroxyvitamin D3. VDR(-/-) mice exhibited significantly higher bacterial loading than wild-type VDR(+/+) mice (P<0.01) and cleared the chlamydial infection in 39 days, compared with 18 days for VDR(+/+) mice. Monocytes and neutrophils were more numerous in the uterus and oviduct of VDR(-/-) mice than in VDR(+/+) mice (P<0.05) at d 45 after infection. Pre-treatment of HeLa cells with 10nM or 100nM 1,25-dihydroxyvitamin D3 decreased the infectivity of C. muridarum (P<0.001). Several differentially expressed protein spots were detected by proteomic analysis of chlamydial-infected HeLa cells pre-treated with 1,25-dihydroxyvitamin D3. Leukocyte elastase inhibitor (LEI), an anti-inflammatory protein, was up-regulated. Expression of LEI in the ovary and oviduct of infected VDR(+/+) mice was greater than that of infected VDR(-/-) mice. We conclude that the vitamin D endocrine system reduces the risk for prolonged chlamydial infections through regulation of several proteins and that LEI is involved in its anti-inflammatory activity.

Effect of topical vaginal products on the detection of prostate-specific antigen, a biomarker of semen exposure, using ABAcards.

BACKGROUND: Prostate-specific antigen (PSA) is a biomarker of recent semen exposure. There is currently only limited information on whether topical vaginal products affect PSA assays. We investigated this question using various dilutions of several vaginal products (lubricants and spermicides) and the Abacus ABAcard for PSA detection. STUDY DESIGN: Pooled semen controls and various dilutions of nonoxynol-9 (N9), carboxymethyl cellulose (CMC), Replens, Gynol 2, K-Y jelly, Astroglide, Surgilube, combined with pooled semen dilutions, were tested for PSA using the Abacus ABAcard. RESULTS: N9 (2% with saline) and CMC did not appear to affect the results of testing with the ABAcard, but not all semen dilutions were tested. The other products (including Replens and Gynol, which is 2% N9 with propylene glycol, K-Y, Astroglide and Surgilube) at some of the dilutions tested either affected or gave invalid results with PSA testing using the ABAcard. Both Gynol 2 and K-Y at 1:10 dilution gave false-positive results. CONCLUSIONS: Some vaginal products affect PSA results obtained by using the semiquantitative ABAcard. In vivo confirmation is necessary to further optimize PSA detection when topical vaginal products are present.

Anogenital human papillomavirus in sexually abused and nonabused children: a multicenter study.

OBJECTIVES: To characterize the epidemiology of genital human papillomavirus (HPV) infection in children without previous consensual sexual activity, comparing HPV prevalence by certainty of child sexual abuse (CSA). PATIENTS AND METHODS: Patients presenting for evaluation of CSA in 8 sites in Atlanta, Houston, Harrisburg, and New York City were recruited along with patients presenting for unrelated health visits. CSA certainty was classified as definite, probable, possible, or no evidence following published guidelines and the results of history, physical examination, and laboratory tests. Urine and swabs of external genitalia were tested for HPV using L1 consensus polymerase chain reaction. RESULTS: The study included 576 participants (89.9% female) aged 6 months to 13 years (mean: 7.9); 534 of whom were evaluated for CSA and 42 for unrelated reasons. Of those evaluated for CSA, 14 had genital warts. One or more HPV types were detected in 11.8% (61 of 517) of participants with adequate samples. HPV detection was more likely among abused participants (definite, probable, or possible) than among participants without evidence of CSA (13.7% and 1.3%, respectively; P < .0001) and increased with certainty of abuse (8.4%, 15.6%, and 14.5% in participants with possible, probable, and definite CSA, respectively; P < .0001). Participants aged 10 years or older had a higher prevalence of HPV (20.6%) than others (5.6%) (P < .0001). CSA, anogenital warts, and age were independently associated with HPV detection. CONCLUSIONS: HPV detection was associated with CSA and increased with CSA certainty. In this population, genital HPV seemed to behave as a sexually transmitted infection.

Effect of specimen order on Chlamydia trachomatis and Neisseria gonorrhoeae test performance and adequacy of Papanicolaou smear.

STUDY OBJECTIVE: To evaluate the effect of specimen collection order on the performance of diagnostic tests for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC), and the specimen adequacy of ThinPrep Papanicolaou (Pap) smears. DESIGN: Prospective cohort study. SETTING: Public adolescent clinic. POPULATION: 313 women. INTERVENTIONS: The order of five cervical testing specimens was randomized for (1) ligase chain reaction (LCR) and (2) polymerase chain reaction (PCR) for both CT and GC; (3) transcription-mediated amplification (TMA) for CT; (4) culture for CT; and (5) cytology and LCR for GC and CT performed on ThinPrep Pap specimens. For CT and GC, a reference standard was based on three different tests performed on separate specimens. Generalized estimating equations were used to account for repeated measures. MAIN OUTCOME MEASURE: Sensitivity and specificity of diagnostic tests. RESULTS: The proportion of inadequate Pap smears was independent of specimen order. As a group, nucleic acid amplification test (NAAT) sensitivity and specificity for GC and CT were similar in the first two (early) and last three (late) swabs. Although point estimates for sensitivity were higher in the early swabs compared to the late swabs for GC LCR (13% difference), GC PCR (13%), and CT TMA (10%), these differences were not statistically significant. Their clinical significance warrants further investigation. CONCLUSIONS: In clinical settings where both Pap smears and STI testing are performed in adolescents, clinical considerations can influence the order of specimen collection, since neither Pap specimen adequacy nor test performance of NAAT for CT and GC were significantly associated with swab order.

Association of Chlamydia trachomatis with persistence of high-risk types of human papillomavirus in a cohort of female adolescents.

Human papillomavirus (HPV) infection is a necessary but not sufficient cause of cervical cancer. While chlamydia infection has been associated with cervical cancer, the meaning of this association remains unclear. The authors' objective was to investigate this association by evaluating whether concurrent genital tract infections are associated with HPV persistence, a precursor to cervical cancer. Interview data and biologic samples for HPV, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and bacterial vaginosis testing were collected from female adolescents in an Atlanta, Georgia, longitudinal cohort study at 6-month visits (1999-2003). Associations with persistence (detection of the same HPV type at two sequential visits (visit pair)) were assessed among subjects with 2-5 visits and > or =6 months of follow-up. Associations were evaluated by logistic regression using methods for correlated data. Type-specific persistence of high-risk HPV types was detected in 77 of 181 (43%) analyzed visit pairs. Concurrent infection with C. trachomatis was independently associated with persistence of high-risk HPV types (adjusted odds ratio = 2.1, 95% confidence interval: 1.0, 4.1). Infection with more than one HPV type at the initial visit was also associated with high-risk persistence (adjusted odds ratio = 2.8, 95% confidence interval: 1.6, 4.9). The association between chlamydia infection and cervical cancer may be due to an effect of chlamydia infection on persistence of high-risk HPV.

Host inflammatory response and development of complications of Chlamydia trachomatis genital infection in CCR5-deficient mice and subfertile women with the CCR5delta32 gene deletion.

T cell immunity protects against diseases caused by the obligate intracellular bacterium Chlamydia trachomatis. Incidentally, host inflammatory response that includes T cells appears to also contribute to the pathogenesis of chlamydial diseases such as trachoma and tubal factor infertility (TFI). Therefore, designing effective prevention strategies requires a delineation of immune processes responsible for pathology and those mediating immunity, and identification of the immunogenetic factors predisposing to complication development. The chemokine receptor CCR5 is crucial for T cell activation and function since its deficiency causes suppression of T cell response. We investigated the hypothesis that the clearance of genital chlamydial infection in CCR5-deficient mice could be delayed in the short term; however, a beneficial effect could include protection against inflammation-related complications such as TFI. In a translational study in humans, we investigated the effect of a functional 32 bp deletion in the CCR5 gene on the risk of developing tubal pathology in Dutch Caucasian women with immunologic evidence [i.e., immunoglobulin G (IgG) responses] of chlamydial infection. When genitally-infected wild-type (WT) and CCR5 knockout (CCR5KO) mice were evaluated for microbiologic shedding of chlamydiae, there was a greater intensity of infection and delayed resolution in the knockout mice. However, compared to WT mice, the fertility of infected CCR5KO mice (measured by pregnancy rate) was only mildly affected in the short term and unaffected in the long term (70% vs 30% reduction in the short term, and 50 vs 0% in the long term, respectively). Immunobiologic analysis revealed that the diminished capacity of CCR5KO to control acute chlamydial infection correlated with the relatively low chemokine [interferon-inducible protein 10 (IP-10) and regulated upon activation normal cell expressed and secreted (RANTES)] and cytokine (mainly interferon-gamma and tumor necrosis factor-alpha) expression corresponding to a poor early T-helper I response. However, the reduced incidence of complications in the CCR5KO mice appears to correlate with the low activity of long term inflammatory mediators. Besides, the translational studies in humans revealed that among patients with positive anti-chlamydial IgG responses, tubal pathology correlated with a low incidence of CCR5delta32 deletion (7%), while women without tubal pathology had higher incidence of the CCR5delta32 deletion (31%) as compared to controls (19%). Thus, in mice and humans the inflammation associated with CCR5 function may predispose to development of complications of chlamydial infection, such as TFI.

Epidemiology of human papillomavirus infection and abnormal cytologic test results in an urban adolescent population.

We determined the prevalence of and the risk factors for human papillomavirus (HPV) infection and abnormal cytologic test results in 312 adolescent girls (mean age, 16.1 years). Subjects had a median of 2 years of sexual activity and 4 lifetime sex partners. Cervical HPV was detected by use of L1-consensus polymerase chain reaction in 64% of subjects; half of those with HPV had >1 type, and 77% had >/=1 high-risk type. Independent risk factors for HPV were lifetime number of sex partners, age of partner, and douching. Cytologic abnormalities were common (20.9% of subjects had atypical squamous cells of uncertain significance, and 17.0% had high- or low-grade squamous intraepithelial lesions) and were significantly associated with detection of HPV (P=.0001); however, most (51.6%) subjects with HPV had normal cytologic test results.

Comparison of methods for detection of Chlamydia trachomatis and Neisseria gonorrhoeae using commercially available nucleic acid amplification tests and a liquid pap smear medium.

Annual screening for Chlamydia trachomatis infection is currently recommended for sexually active women 15 to 25 years old and for women older than 25 if they have a new or multiple sex partners and have not used condoms during the previous 3 months. Annual screening for cervical abnormalities using the Pap smear has achieved a substantial reduction in morbidity and mortality from cervical cancer. Screening for Neisseria gonorrhoeae infection has likely contributed significantly to the reduction in the rates of gonococcal infection. The introduction of liquid Pap smear methods using exfoliated cervical cells presents an opportunity to screen for these three conditions using one specimen. We evaluated the preservation of C. trachomatis and Neisseria gonorrhoeae DNAs from ThinPrep liquid media (PreservCyt; Cytyc Corp., Boxborough, Mass.); tested the feasibility of using a clinical specimen of this medium for the detection of cytologic abnormalities, C. trachomatis, and N. gonorrhoeae; evaluated the agreement between ligase chain reaction (LCR) performed on PreservCyt and LCR performed on a cervical specimen; and compared the performance of LCR performed on PreservCyt to those of LCR performed on a cervical specimen, culture, PCR performed on a cervical specimen, on urine, and on a vaginal specimen (a multiple-site infection status standard), and transcription-mediated amplification (for C. trachomatis only) from 255 sexually active adolescent women. The agreement between LCR performed on PreservCyt and LCR from a cervical swab in LCx transport medium was high (for C. trachomatis, agreement = 0.97 and kappa = 0.92; for N. gonorrhoeae, agreement = 0.99 and kappa = 0.96). Test performances were similar for LCR-urine, LCR-cervix, and LCR-ThinPrep, with sensitivities from 93 to 99% for C. trachomatis and 81 to 83% for N. gonorrhoeae and specificities from 95.5 to 99% for C. trachomatis and 99.1 to 99.6% for N. gonorrhoeae using a PCR-based multiple-site infection status standard. This is the first study to examine the agreement between liquid cytologic media and multiple nucleic acid amplification tests for the detection of C. trachomatis and N. gonorrhoeae from patient samples. Cytologic fluid shows promise for simultaneous screening for cytologic abnormalities and sexually transmitted infections.