Lisa A. Cosentino

Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study.

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1β and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1β and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal−Wallis analysis of variance with Dunn’s multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1β determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.

Genital Tract Shedding of Herpes Simplex Virus Type 2 in Women: Effects of Hormonal Contraception, Bacterial Vaginosis, and Vaginal Group B Streptococcus Colonization

BACKGROUND Genital infections due to herpes simplex virus type 2 (HSV-2) are characterized by frequent reactivation and shedding of the virus and by the attendant risk of transmission to sexual partners. We investigated the effects of vaginal coinfections and hormonal contraceptive use on genital tract shedding of HSV-2 in women. METHODS A total of 330 HSV-2-seropositive women were followed every 4 months for a year. At each visit, one vaginal swab specimen was obtained for detection of HSV-2 by polymerase chain reaction, a second vaginal swab specimen was obtained for detection of group B Streptococcus (GBS) organisms and yeast by culture, and a vaginal smear was obtained for the diagnosis of bacterial vaginosis by Gram staining. RESULTS HSV-2 DNA was detected in 88 (9%) of 956 vaginal swab specimens. Independent predictors of genital tract shedding of HSV-2 were HSV-2 seroconversion during the previous 4 months (adjusted odds ratio [aOR], 3.0; 95% confidence interval [CI], 1.3-6.8), bacterial vaginosis (aOR, 2.3; 95% CI, 1.3-4.0), high-density vaginal GBS colonization (aOR, 2.2; 95% CI, 1.3-3.8), and use of hormonal contraceptives (aOR, 1.8; 95% CI, 1.1-2.8). CONCLUSIONS The present study identifies hormonal contraceptive use, bacterial vaginosis, and high-density vaginal GBS colonization as risk factors for genital tract shedding of HSV-2 in women. Because hormonal contraceptives are used by millions of women worldwide and because bacterial vaginosis and vaginal GBS colonization are common vaginal conditions, even modest associations with HSV-2 shedding would result in substantial attributable risks for transmission of the virus.

The Associations Between Pelvic Inflammatory Disease, Trichomonas vaginalis Infection, and Positive Herpes Simplex Virus Type 2 Serology

Objective: Roles for Chlamydia trachomatis and Neisseria gonorrhoeae infections in pelvic inflammatory disease pathogenesis are well delineated; however, the etiologic contributions of herpes simplex virus type 2 (HSV-2) and Trichomonas vaginalis have been underexplored. Goal: The goal of this study was to investigate the association between acute and plasma cell endometritis, fallopian tube obstruction, HSV-2 serology, and T. vaginalis infection. Study Design: The authors conducted a cross-sectional secondary analysis of 736 women at risk for bacterial sexually transmitted diseases that used endometrial biopsy data obtained at enrollment as well as hysterosalpingography results obtained 12 weeks after enrollment. Results: Women diagnosed with T. vaginalis at enrollment were more likely to have histologic evidence of acute endometritis. Both plasma cell and acute endometritis were significantly more common among women with positive serology HSV-2; furthermore, women coinfected with HSV-2 and C. trachomatis, N. gonorrhoeae, T. vaginalis, or bacterial vaginosis were much more likely to be diagnosed with acute endometritis than were women infected with HSV-2 or one of these pathogens alone. Among women with available HSV-2 serology and hysterosalpingogram results, HSV-2 was the only genital tract pathogen infection associated with fallopian tube obstruction. Conclusions: Our analyses demonstrate that T. vaginalis infection and positive HSV-2 serology are associated with endometritis. Further work will be needed to determine the specific roles these pathogens may play in pelvic inflammatory disease pathogenesis.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Strand Displacement Amplification and Relevance of the Amplification Control for Use with Vaginal Swab Specimens

ABSTRACT Vaginal swab specimens may be preferable to cervical swab or urine specimens for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae because of the ease of specimen collection and transport. The purpose of this study was to evaluate whether vaginal swab specimens are equivalent to cervical swab specimens for the detection of N. gonorrhoeae and C. trachomatis by the Becton Dickinson strand displacement amplification assay (SDA) with the BDProbeTec ET instrument and then to evaluate the use of the amplification control in a clinical research setting. In the first phase, vaginal and cervical swab specimens were obtained from 455 symptomatic women aged 18 to 40 attending primary health care and sexually transmitted disease clinics. Thirty-nine specimens (8.6%) had true-positive results for N. gonorrhoeae and 37 specimens (8.1%) had true-positive results for C. trachomatis. The sensitivity of SDA was superior to that of culture for the detection of N. gonorrhoeae with vaginal swab specimens and equivalent to that of the Roche PCR for the detection of C. trachomatis with cervical swab specimens. In the second phase of the study, 1,411 consecutively collected vaginal swab specimens were evaluated, with 357 (25.3%) specimens giving indeterminate readings on the basis of the result for the amplification control. The prevalences of sexually transmitted pathogens in vaginal swab specimens with and without use of the amplification control were 6.0 and 5.8%, respectively, for C. trachomatis and 3.1 and 3.0%, respectively, for N. gonorrhoeae. Although, vaginal swab specimens were equivalent to cervical swab specimens for the detection of N. gonorrhoeae and C. trachomatis by SDA with respect to sensitivity, one in four vaginal swab specimens yielded an indeterminate result when the amplification control was used. The amplification control has limited value for use with vaginal swab specimens.

Cytokines, Pregnancy, and Bacterial Vaginosis: Comparison of Levels of Cervical Cytokines in Pregnant and Nonpregnant Women with Bacterial Vaginosis

BACKGROUND Pregnancy has been considered to be a time of relative immune compromise. Lower-genital-tract immune response appears to be influenced by pregnancy. The objective of this study was to compare, in pregnant versus nonpregnant women, endocervical proinflammatory-cytokine expression in response to bacterial vaginosis. METHODS Endocervical levels of interleukin (IL)-1 beta , IL-6, and IL-8 in 99 pregnant and 99 nonpregnant women, all with bacterial vaginosis and without concurrent sexually transmitted infections, were assessed by ELISA. Vaginal flora was characterized on the basis of quantitative vaginal cultures. RESULTS Women in the 2 groups differed with respect to smoking status and microbiological constituents responsible for bacterial vaginosis. When the data were stratified by these potential confounders, the levels of all 3 proinflammatory endocervical cytokines were significantly higher in pregnant women than in nonpregnant women. CONCLUSIONS The proinflammatory cytokine milieu in the cervix is enhanced in pregnant women with bacterial vaginosis, compared with that in nonpregnant women. The notion of pregnancy as an immune-compromised state may be anatomically compartment specific.

Use of Nucleic Acid Amplification Testing for Diagnosis of Anorectal Sexually Transmitted Infections

ABSTRACT Nucleic acid amplification testing (NAAT) has become the preferred method to detect Chlamydia trachomatis and Neisseria gonorrhoeae, but no commercial tests are cleared by the U.S. Food and Drug Administration for use with rectal swab samples. This study evaluated the performance of strand displacement amplification (SDA) and transcription-mediated amplification (TMA) to detect C. trachomatis and N. gonorrhoeae and to determine if TMA could also detect Mycoplasma genitalium and Trichomonas vaginalis in men and women reporting a history of receptive anal intercourse. Discordant results between the NAATs were reevaluated using the Aptima CT or Aptima GC assay, each of which targets primers other than those targeted by the Aptima Combo 2 (AC2) assay, as the confirmatory test. Of 497 evaluable participants, 41 (8.2%) were positive for C. trachomatis, 21 (4.2%) were positive for N. gonorrhoeae, 26 (5.2%) were positive for T. vaginalis, and 47 (9.5%) were positive for M. genitalium. The sensitivity and specificity of the C. trachomatis test were 100% and 99.8% for AC2 and 56.1% and 100% for SDA, respectively. The sensitivity and specificity of the N. gonorrhoeae test were 100% and 100% for AC2 and 76.2% and 100% for SDA, respectively, while culture was only 23.8% sensitive. Of the 114 participants who had a positive result for any of the four infectious agents, 16 were positive for two pathogens and 3 were positive for three pathogens. These data suggest that rectal infection is common and that the AC2 is superior to SDA for the detection of C. trachomatis and N. gonorrhoeae from rectal swab samples.

Hormonal contraceptive use modulates the local inflammatory response to bacterial vaginosis

Objectives: To compare cervical concentrations of numerous cytokines/chemokines in women with bacterial vaginosis (BV) compared with the levels detected after BV resolution and determine if hormonal contraceptive use modulates the local inflammatory response to BV. Methods: Cervical secretions from 81 women with BV at enrollment and normal flora at one-month follow-up were analysed for 10 different cytokines/chemokines using multiplexed fluorescent bead-based immunoassays. Results: BV was associated with significantly higher concentrations of IL-1β, tumour necrosis factor (TNF), interferon-γ, IL-2, IL-4, and IL-10 compared with the levels detected in the presence of normal vaginal flora. Analysis of results stratified by contraceptive practice demonstrated significantly lower levels of numerous cytokines among women with BV using hormonal contraceptives compared with those women with BV not using hormonal contraceptives. Hormonal contraceptive use was also associated with a statistically significant lesser change in TNF levels between the two study visits compared with the amount of change detected between visits among women who denied their use. Conclusions: Despite increases in the levels of both pro and anti-inflammatory cytokines in the lower genital tract of women with BV, the overall balance of these two types of molecules was maintained. The character of this local inflammatory response may help explain the typical absence of overt signs of inflammation among women with BV. In addition, hormonal contraceptive use was associated with significantly lower levels of the pro-inflammatory molecules TNF, interferon-γ, and granulocyte macrophage colony-stimulating factor in women with BV, but did not significantly reduce the levels of IL-10, a key anti-inflammatory cytokine. These results suggest the possibility of an association between hormonal contraceptive use and altered genital tract immunity.

Patterns of Extragenital Chlamydia and Gonorrhea in Women and Men Who Have Sex With Men Reporting a History of Receptive Anal Intercourse.

Background Screening for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) in men who have sex with men is risk based. Despite high frequencies of oral and receptive anal intercourse (RAI) among women, extragenital screening is not recommended. Methods Women (n = 175) and men who have sex with men (n = 224) primarily recruited from a sexually transmitted infection clinic reporting a lifetime history of RAI completed a structured questionnaire and clinician-collected swab samples from the rectum, pharynx, vagina (women), and urine (men). CT and GC were detected using 2 commercial nucleic acid amplification tests (Aptima Combo 2; Hologic, Inc, Bedford, MA; Xpert CT/NG, Cepheid Innovation, Sunnyvale, CA). Results The median age of the population was 26 years, 62% were white, and 88% were enrolled from a sexually transmitted disease clinic. Men were more likely than women to have GC (22.8% vs. 3.4%) and CT (21.9% vs. 12.6%). In men versus women, GC was detected in 16.5% versus 2.3% of pharyngeal swabs, 11.6% versus 2.3% of rectal swabs, and 5.4% versus 2.9% of urine samples or vaginal swabs. C. trachomatis was detected in 2.2% versus 1.7% of pharyngeal swabs, 17.4% versus 11.4% of rectal swabs, and 4.5% versus 10.3% for urogenital sites in men versus women. Overall 79.6% of CT and 76.5% of GC in men and 18.2% of CT and 16.7% of GC in women were detected only in the pharynx or rectum. Conclusion Reliance on urogenital screening alone misses most of GC and CT in men and more than 15% of infections in women reporting RAI.

Interlaboratory reproducibility of female genital tract cytokine measurements by Luminex: Implications for microbicide safety studies

The interlaboratory reproducibility of cytokine measurements from cervicovaginal samples by Luminex has not been reported. Using cervicovaginal lavage specimens collected on three study days from 12 women participating in a Phase I microbicide study, we measured a panel of eight cytokines in three independent laboratories. Four (IFN-γ, IL-10, IL-17, and TNF) were below the limit of detection in the majority (85%) of samples in either two or all three laboratories, an observation that may guide analyte selection for future studies. Good interlaboratory agreement (intraclass correlation coefficient, r>0.7) in absolute levels was observed for IL-1β, IL-6, and IL-8, while poor agreement was seen for IFN-α2 (r=0.47). When considering within-subject change from baseline (pre-product, at study-day 0) to either post-product visit (study-days 7 and 14), IL-1β and IL-6 exhibited good interlaboratory agreement (r>0.7), while IFN-α2 and IL-8 did not. Future studies addressing the clinical utility of specific biomarkers of inflammation for microbicide trials should consider reproducibility in the context of defining biologically meaningful thresholds of change for candidate biomarkers, ensuring that such change can be reliably distinguished from background variability.

Systemic immune response to Trichomonas vaginalis infection during pregnancy.

Objective: The objective of this study was to characterize the systemic immune response in women with trichomoniasis in pregnancy as compared with uninfected women. Study Design: A nested case control study was performed on 195 serum samples. Serum concentrations of cytokines, chemokines, and C-reactive protein (CRP) were compared between infected and uninfected women. Cytokines and chemokines were measured using a multiplex bead assay. The CRP concentrations were determined using a standard enzyme-linked immunosorbent assay method. Results: The median serum concentration of granulocyte–macrophage colony-stimulating factor (GM-CSF) was significantly higher in the trichomonas-infected group compared with the uninfected group (8.9 pg/mL vs. 5.7 pg/mL; P <0.001). The mean log-transformed CRP values were higher in the infected group compared with the uninfected group (1.66 vs. 1.27; P = 0.03). Conclusions: The results of this study suggest that trichomoniasis during pregnancy can lead to a systemic immune response in some women as exhibited by elevation in the serum concentrations of both GM-CSF and CRP.