Cervicovaginal Lavage Research Articles

Cervicovaginal HIV-1 Shedding in Women Taking Antiretroviral Therapy in Burkina Faso

Background:Antiretroviral therapy (ART) reduces transmission of HIV-1. However, genital HIV-1 can be detected in patients on ART. We analyzed factors associated with genital HIV-1 shedding among high-risk women on ART in Burkina Faso.Methods:Plasma viral load (PVL) and enriched cervicovaginal lavage HIV-1 RNA were measured every 3–6 months for up to 8 years. Random-effects logistic and linear regression models were used to analyze associations of frequency and quantity of genital HIV-1 RNA with behavioral and biological factors, adjusting for within-woman correlation. The lower limit of detection of HIV-1 RNA in plasma and eCVL samples was 300 copies per milliliter.Results:One hundred and eighty-eight participants initiated ART from 2004 to 2011. PVL was detectable in 16% (171/1050) of visits, in 52% (90/174) of women. Cervicovaginal HIV-1 RNA was detectable in 16% (128/798) of visits with undetectable plasma HIV-1 RNA in 45% (77/170) of women. After adjusting for PVL, detectable cervicovaginal HIV-1 RNA was independently associated with abnormal vaginal discharge and use of nevirapine or zidovudine vs. efavirenz and stavudine, respectively; longer time on ART and hormonal contraception were not associated with increased shedding. The presence of bacterial vaginosis, herpes simplex virus-2 DNA, and the use of nevirapine vs efavirenz were independently associated with an increased quantity of cervicovaginal HIV-1 RNA.Conclusions:Certain ART regimens, abnormal vaginal discharge, bacterial vaginosis, and genital herpes simplex virus-2 are associated with HIV-1 cervicovaginal shedding or quantity in women on ART after adjusting for PVL. This may reduce the effectiveness of ART as prevention in high-risk populations.

Optimizing Viable Leukocyte Sampling from the Female Genital Tract for Clinical Trials: An International Multi-Site Study

BackgroundFunctional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear.Methods and FindingsWe enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4+ T cells in the female genital tract express the α4β7 integrin, an HIV envelope-binding mucosal homing receptor.ConclusionsCVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.

Increased Exosomal MicroRNA-21 and MicroRNA-146a Levels in the Cervicovaginal Lavage Specimens of Patients with Cervical Cancer

Well-run screening programs for cervical cancer in the population at risk have been shown to result in a sharp decrease in the incidence and mortality of cervical cancer in a number of large populations. Expression patterns of a recently identified biomarker family, microRNA, appear to be characteristic of tumor type and developmental origin. Several tumors have been reported to actively release exosomes carrying microRNAs. The present study has determined the association of microRNAs with cervical cancer-derived exosomes. The cervical cancer-derived exosomes were enriched in the cervicovaginal lavages specimens and the abundance of exosomes and exosomal microRNAs was detected by electron microscopy, western blot analysis, RT-qPCR and microRNA target reporter vector. The microRNA-21 and microRNA-146a, which were up-regulated in cervical cancer patients, were associated with the high levels of cervical cancer-derived exosomes. In conclusion, we demonstrated the abundance of exosomes in the cervicovaginal lavage specimens of women with cervical cancer. Furthermore, our results indicated that abnormally high levels of microRNA-21 and microRNA-146a existed in the cervical cancer-derived exosomes and the two microRNAs were functional in 293T cells.

Unbiased Proteomics Analysis Demonstrates Significant Variability in Mucosal Immune Factor Expression Depending on the Site and Method of Collection

Female genital tract secretions are commonly sampled by lavage of the ectocervix and vaginal vault or via a sponge inserted into the endocervix for evaluating inflammation status and immune factors critical for HIV microbicide and vaccine studies. This study uses a proteomics approach to comprehensively compare the efficacy of these methods, which sample from different compartments of the female genital tract, for the collection of immune factors. Matching sponge and lavage samples were collected from 10 healthy women and were analyzed by tandem mass spectrometry. Data was analyzed by a combination of differential protein expression analysis, hierarchical clustering and pathway analysis. Of the 385 proteins identified, endocervical sponge samples collected nearly twice as many unique proteins as cervicovaginal lavage (111 vs. 61) with 55% of proteins common to both (213). Each method/site identified 73 unique proteins that have roles in host immunity according to their gene ontology. Sponge samples enriched for specific inflammation pathways including acute phase response proteins (p = 3.37×10−24) and LXR/RXR immune activation pathways (p = 8.82×10−22) while the role IL-17A in psoriasis pathway (p = 5.98×10−4) and the complement system pathway (p = 3.91×10−3) were enriched in lavage samples. Many host defense factors were differentially enriched (p<0.05) between sites including known/potential antimicrobial factors (n = 21), S100 proteins (n = 9), and immune regulatory factors such as serpins (n = 7). Immunoglobulins (n = 6) were collected at comparable levels in abundance in each site although 25% of those identified were unique to sponge samples. This study demonstrates significant differences in types and quantities of immune factors and inflammation pathways collected by each sampling technique. Therefore, clinical studies that measure mucosal immune activation or factors assessing HIV transmission should utilize both collection methods to obtain the greatest representation of immune factors secreted into the female genital tract.

Effect of depot medroxyprogesterone acetate on human β-defensin production and structural integrity of the human vaginal epithelium

BackgroundRecent studies implicate the use of hormonal contraceptives, particularly depot medroxyprogesterone acetate (DMPA; Depo-Provera), with increased risk of HIV-1 acquisition. In this study, we recruited long-term users of hormonal contraceptives to further understand the biological mechanism behind this phenomenon. Methods83 healthy, premenopausal women using DMPA (22 participants), combined oral contraceptives (17), NuvaRing (17), or non-hormonal contraceptives (26) were recruited in Birmingham, AL, USA, for a vaginal biopsy, blood draw, and cervicovaginal lavage (CVL). Participants were excluded if they were pregnant or symptomatic for vaginitis or ulcers. Informed consent was obtained from all patients; the study was approved by the Institutional Review Board of University of Alabama at Birmingham. Vaginal epithelium was assessed with two distinct measurements: a mean thickness and shortest distance (from the lumen to the nearest lamina propria projection; minimum 100 points per biopsy). CVL antiviral content was assessed with ELISA and Luminex. RNA sequencing was performed on four DMPA and four control vaginal biopsies with use of laser capture microdissection to dissect the epithelium from the lamina propria. FindingsDMPA use was significantly associated with thinner vaginal epithelium (median DMPA 290·7 μm; median control 353·8 μm; p=0·01). DMPA and NuvaRing suppressed production of human β defensin-2 (HBD2) (control 13 198 pg/mL; DMPA 741·5 pg/mL, p=0·0002; NuvaRing 326·7 pg/mL, p=0·0003) and HBD3 (control 776·4 pg/mL; DMPA 50·2 pg/mL, p<0·0001; NuvaRing 41·9 pg/mL, p=0·0004) in CVL. HBD3 transcript levels were 330-fold reduced in the vaginal epithelium of DMPA users compared with controls (control fragments per kilobase of exon per million fragments mapped [FPKM] 69·7, DMPA FPKM 0·21; p=4·17 × 10−5; q=8·08 × 10−3). Immunofluorescence staining on DMPA biopsies confirmed the loss of HBD3 protein. DMPA users showed significant decreases in key structural genes including corneodesmosin (control FPKM 9·9; DMPA FPKM 0·26; p=1·36 × 10−6; q=5·86 × 10−4) and loricrin (control FPKM 10·2; DMPA FPKM 0·9; p=1·3 × 10−7; q=1·15 × 10−4). Markers of wound healing, including haemoglobin α2 (control FPKM 3·8; DMPA FPKM 40; p=1·6 × 10−5; q=3·7 × 10−3) and plasminogen activator (control FPKM 0·9; DMPA FPKM 12·7; p=2·4 × 10−8; q=2·8 × 10−5), were significantly upregulated in the vaginal epithelium of DMPA users compared with controls. InterpretationBecause HBDs are implicated in a specific anti-HIV response, loss of HBDs in the genital tract could partly account for the increased risk of infection in DMPA users. Decreased transcription of structural genes combined with increased expression of genes indicating wound healing suggests that DMPA weakens the natural barrier of the vaginal epithelium, which could lead to increased epithelial damage and exposure of target cells to mucosal pathogens such as HIV-1. FundingThis work was supported by NIH Grant P01 AI083027.

Association of plasma viremia with HIV-1 RNA levels in cervicovaginal lavage secretions in HIV-1 seropositive ART naïve women from Pune, India

BackgroundCoherent drug/microbicide/vaccine development research would benefit through a precise knowledge of HIV dissemination and its persistence in the female genital tract. Understanding relationship between plasma viremia and cervicovaginal HIV shedding may help to unveil mechanisms underlying transmission, compartmentalization and pathogenesis. ObjectivesTo study the association between HIV-1 RNA levels in the plasma and CVL specimens. Study designWhole blood, plasma and CVL specimens were collected from 36 ART naïve HIV-1 seropositive women qualifying the study inclusion criteria. Absolute CD4 counts, plasma and CVL HIV-1 RNA levels were estimated using commercially available kits (BD MultiSET™ Kit, Becton Dickinson, US and Abbott RT, Abbott Molecular, Germany). Correlation between plasma and CVL viral load was estimated by the Spearman's rank correlation coefficient. Additionally, the correlation between CVL viral load and absolute CD4 counts was studied. ResultsHIV-1 viral load in the CVL specimens was successfully quantified using the Abbott RT. Twenty-seven of 36 women (75%) had detectable HIV-1 RNA levels in plasma and CVL specimens. The CVL viral load did not show any correlation with plasma viral load (ρ=0.281, p=0.096) and showed a ‘moderate correlation’ (ρ=−0.563, p=0.0004) with absolute CD4 counts. ConclusionsAlbeit, the Abbott RT is designed for estimating plasma HIV-1 RNA levels, the study reports its use for estimating HIV-1 RNA levels in the CVL specimens as well. In accordance with the previous studies, our results suggest that plasma and CVL viral load are not correlated and plasma viremia might not solely predict cervico vaginal HIV shedding.

Novel preclinical models of topical PrEP pharmacodynamics provide rationale for combination of drugs with complementary properties

BackgroundThe limited success of recent HIV topical pre-exposure prophylaxis clinical trials highlights the need for more predictive models of drug efficacy that better simulate what may happen during sexual exposure. To address this gap, we developed complementary in vitro models to evaluate the ability of drugs to retain anti-HIV activity if cells were washed with seminal plasma (simulating what may happen following exposure to ejaculate), and to protect drug-naive T cells (representing newly recruited immune cells) co-cultured with explants that had been pretreated with drug. We focused on tenofovir disoproxil fumarate (TDF), the non-nucleoside reverse transcriptase inhibitors dapivirine (DPV) and IQP-0528, and the entry inhibitors maraviroc (MVC) and the D-peptide chol-PIE-12 trimer (PIE12). Studies were extended to macaques and the ability of cervical biopsies obtained from animals treated with an intravaginal ring formulation of IQP-0528 to protect ex vivo co-cultured T cells was determined. The antiviral activity of cervicovaginal lavage samples against a primary Clade C isolate was also measured and correlated with drug levels.ResultsCells exposed to TDF were equally protected from HIV whether or not the drug-treated cells were washed with medium or seminal plasma prior to challenge. In contrast, several-fold higher concentrations of NNRTIs and entry inhibitors were needed to attain similar levels of HIV inhibition following a wash with seminal plasma. Conversely, the NNRTIs and PIE12, but not TDF or MVC, were effectively transferred from ex vivo treated explants and protected co-cultured T cells. Biopsies obtained from IQP-0528 ring-treated macaques also protected co-cultured T cells with viral inhibition ranging from 42-72%. Antiviral activity correlated with the concentration of drug recovered. Combinations of TDF with IQP-0528 protected in both in vitro models.ConclusionsTogether, these models suggest that intracellularly retained drugs such as TDF may protect resident immune cells following coitus but sustained delivery may be required to protect immune cells subsequently recruited into the genital tract. Sustained delivery may also be critical for NNRTIs, which are rapidly transported out of cells and could be lost following sexual intercourse. An ideal approach may be a combination of drugs with complementary bioavailability profiles formulated for sustained delivery.

HIV-1 outcompetes HIV-2 in dually infected Senegalese individuals with low CD4⁺ cell counts.

Dual infection with HIV-1 and HIV-2, which is not uncommon in West Africa, has implications for transmission, progression, and antiretroviral therapy (ART). Few studies have examined viral dynamics in this setting. Our objective was to directly compare HIV-1 and HIV-2 viral loads and to examine whether this relationship is associated with CD4⁺ cell count. This is a retrospective analysis of data from observational cohort studies. We compared HIV-1 and HIV-2 viral loads from 65 dually infected, ART-naive Senegalese individuals. Participants provided blood, oral fluid, and cervicovaginal lavage (CVL) or semen samples for virologic and immunologic testing. We assessed relationships between HIV-1 and HIV-2 levels using linear regression with generalized estimating equations to account for multiple study visits. After adjusting for CD4⁺ cell count, age, sex, and commercial sex work, HIV-1 RNA levels were significantly higher than HIV-2 levels in semen, CVL, and oral fluids. Despite similar peripheral blood mononuclear cell DNA levels among individuals with CD4⁺ cell counts above 500 cells/μl, individuals with CD4⁺ cell counts below 500 cells/μl had higher HIV-1 and lower HIV-2 DNA levels. Individuals with high CD4⁺ cell counts had higher mean HIV-1 plasma RNA viral loads than HIV-2, with HIV-1 levels significantly higher and HIV-2 levels trending toward lower mean viral loads among individuals with low CD4⁺ cell counts. Our data are consistent with the hypothesis that with disease progression, HIV-1 outcompetes HIV-2 in dually infected individuals. This finding helps explain differences in prevalence and outcomes between HIV-1, HIV-2, and HIV-dual infection.

A multiplexed assay to detect antimicrobial peptides in biological fluids and cell secretions

Mucosal tissues represent the front line in defense against potential pathogens, and one means by which mucosa provide protection is via the secretion of antimicrobials which can interfere with potential pathogens as well as recruit and modify the responses of immune cells. Here we describe adaptation of ELISA assays to microsphere format, facilitating simultaneous quantification of antimicrobial peptides including elafin, MIP3α, HBD2, HBD3, SLPI, RANTES, SDF1, lactoferrin, LL-37, and HNP1-3. The multiplexed assay exhibits excellent reproducibility, shows linearity over a two order of magnitude concentration range for most analytes, is compatible with biological fluids such as cervicovaginal lavage fluid, and presents significant cost and sample savings relative to traditional ELISA assays.

Toward Early Safety Alert Endpoints: Exploring Biomarkers Suggestive of Microbicide Failure

Several microbicides, including nonoxynol-9 (N-9) and cellulose sulfate (CS), looked promising during early trials but failed in efficacy trials. We aimed to identify Phase I mucosal safety endpoints that might explain that failure. In a blinded, randomized, parallel trial, 60 healthy premenopausal sexually abstinent women applied Universal HEC placebo, 6% CS or 4% N-9 gel twice daily for 13½ days. Endpoints included immune biomarkers in cervicovaginal lavage (CVL) and endocervical cytobrushes, inflammatory infiltrates in vaginal biopsies, epithelial integrity by naked eye, colposcopy, and histology, CVL anti-HIV activity, vaginal microflora, pH, and adverse events. Twenty women enrolled per group. Soluble/cellular markers were similar with CS and placebo, except secretory leukocyte protease inhibitor (SLPI) levels decreased in CVL, and CD3(+) and CD45(+) cells increased in biopsies after CS use. Increases in interleukin (IL)-8, IL-1, IL-1RA, and myeloperoxidase (MPO) and decreases in SLPI were significant with N-9. CVL anti-HIV activity was significantly higher during CS use compared to N-9 or placebo. CS users tended to have a higher prevalence of intermediate Nugent score, Escherichia coli, and Enterococcus and fewer gram-negative rods. Most Nugent scores diagnostic for bacterial vaginosis were in N-9 users. All cases of histological inflammation or deep epithelial disruption occurred in N-9 users. While the surfactant N-9 showed obvious biochemical and histological signs of inflammation, more subtle changes, including depression of SLPI, tissue influx of CD45(+) and CD3(+) cells, and subclinical microflora shifts were associated with CS use and may help to explain the clinical failure of nonsurfactant microbicides.

HIV-DNA in the Genital Tract of Women on Long-Term Effective Therapy Is Associated to Residual Viremia and Previous AIDS-Defining Illnesses

ObjectivesTo assess the impact of long-term combined antiretroviral therapy (cART) on HIV-RNA and HIV-DNA levels in cervicovaginal secretions of HIV-1-infected women with sustained undetectable plasma RNA viral load (PVL); to explore factors predictive of residual viral shedding; and to evaluate the risk of heterosexual transmission.MethodsWomen with undetectable PVL (<50 copies/mL) for >6 months were included in this cross-sectional study. HIV-RNA and HIV-DNA were measured in blood and cervicovaginal lavage fluid (CVL). Women were systematically tested for genital infections. The risk of transmission to male partners during unprotected intercourse was estimated.ResultsEighty-one women composed the study population: all had HIV-RNA <40 copies/mL in CVL. HIV-DNA was detectable in CVL of 29/78 patients (37%). There was a weak positive correlation between HIV-DNA levels in PBMCs and CVL (r = 0.20; p = 0.08). In multivariate analysis, two factors were associated with HIV-DNA detection in CVL: previous AIDS-defining illnesses (OR = 11; 95%CI = 2–61) and current residual viremia (20<PVL<50 cp/mL) (OR = 3.4; 95%CI = 1.1–10.9). Neither the classes of cART regimen nor the presence of genital bacterial or fungal colonization were associated with HIV-DNA detection in CVL. Twenty-eight percent of the women had unprotected intercourse with their regular HIV-seronegative male partner, for between 8 and 158 months. None of their male partners became infected, after a total of 14 000 exposures.ConclusionIn our experience, HIV-RNA was undetectable in the genital tract of women with sustained control of PVL on cART. HIV-DNA shedding persisted in about one third of cases, with no substantial evidence of residual infectiousness.

Plasma and Mucosal HIV Viral Loads Are Associated With Genital Tract Inflammation in HIV-Infected Women

Systemic and mucosal inflammation may play a role in HIV control. A cross-sectional comparison was conducted among women in the Women's Interagency HIV Study to explore the hypothesis that compared with HIV-uninfected participants, women with HIV, and, in particular, those with high plasma viral load (PVL) have increased levels of mucosal and systemic inflammatory mediators and impaired mucosal endogenous antimicrobial activity. Nineteen HIV-uninfected, 40 HIV-infected on antiretroviral therapy (ART) with PVL ≤ 2600 copies/mL (low viral load) (HIV-LVL), and 19 HIV-infected on or off ART with PVL >10,000 (high viral load) (HIV-HVL) were evaluated. Immune mediators and viral RNA were quantified in plasma and cervicovaginal lavage (CVL). The CVL antimicrobial activity was also determined. Compared to HIV-uninfected participants, HIV-HVL women had higher levels of mucosal but not systemic proinflammatory cytokines and chemokines, higher Nugent scores, and lower Escherichia coli bactericidal activity. In contrast, there were no significant differences between HIV-LVL and HIV-uninfected controls. After adjusting for PVL, HIV genital tract shedding was significantly associated with higher CVL concentrations of IL-6, IL-1β, MIP-1α, and CCL5 (RANTES) and higher plasma concentrations of MIP-1α. High PVL was associated with higher CVL levels of IL-1β and RANTES, as well as with higher Nugent scores, lower E. coli bactericidal activity, smoking, and lower CD4 counts; smoking and CD4 count retained statistical significance in a multivariate model. Further study is needed to determine if the relationship between mucosal inflammation and PVL is causal and to determine if reducing mucosal inflammation is beneficial.

P1.025 Effects of Abnormal Vaginal Flora and Menopause on Cervicovaginal Fluid Viscosity

<h3>Background</h3> Sexually transmitted pathogens, including HIV, are increased among women having abnormal vaginal flora. The mucus gel layer is a component of innate mucosal immunity, and the presence of mucus may contribute to the viscosity of genital tract fluid. Little is known about the impact of reproductive hormones, menopause and vaginal flora patterns on the viscosity of cervicovaginal fluid. <h3>Methods</h3> Vaginal swabs and cervicovaginal lavage (CVL) were collected from 134 healthy asymptomatic post-menopausal women (n = 23), women in the follicular (n = 26) or proliferative (n = 19) phase, and women using levonogerestrol IUDs (n = 28), DMPA (n = 13) or combined oral contraceptives (n = 25). Vaginal smears were evaluated using the Nugent criteria. The viscosity (centipoise, cP) of each sample was measured in triplicate using a Cambridge MicroSample Viscometer. Student’s t-test, analysis of variance, and linear regression were used to assess statistical significance. <h3>Results</h3> The mean CVL viscosity among the 84 women having a Nugent score &lt; 4 was 1.51(± 0.48) vs. 1.26(± 0.29) cP for the 50 women with abnormal vaginal flora (p = 0.001). There was no difference in CVL viscosity for women with intermediate vs. BV flora (p &gt; 0.99). Similarly, there was no difference in viscosity among women at different phases of their menstrual cycle, nor among women using different hormonal contraceptive methods. However, the CVL of 23 postmenopausal women was less viscous compared to the 111 premenopausal women, 1.16(± 0.26) vs. 1.47(± 0.44) cP respectively (p = 0.001). In a linear regression model, abnormal flora (p = 0.01) and postmenopausal status (p = 0.005) were independently associated with decreased CVL viscosity. <h3>Conclusion</h3> Abnormal flora and being post-menopausal are independently associated with decreased CVL viscosity. Even though phase of menstrual cycle and hormonal contraceptive use has been posited to have an impact on cervical mucus, these data suggest that these factors do not have a measurable impact of vaginal fluid viscosity.

Feasibility and Acceptability of a Novel Cervicovaginal Lavage Self-Sampling Device Among Women in Kigali, Rwanda

The Delphi Screener is a novel cervicovaginal lavage self-sampling device. Sixty women in Kigali (Rwanda) assessed the Screener at 2 consecutive visits. Between the visits, ease of use improved, reported difficulties decreased, and the collected sample weight increased. Most women preferred self-collection over a speculum examination.

Acting locally: innate mucosal immunity in resistance to HIV-1 infection in Kenyan commercial sex workers.

Cohort studies of female commercial sex workers (CSWs) in Kenya were among the first to identify highly HIV-1-exposed seronegative (HESN) individuals. As natural resistance is usually mediated by innate immune mechanisms, we focused on determining whether expression and function of innate signaling pathways were altered locally in the genital mucosa of HESN CSWs. Our results demonstrated that selected pattern-recognition receptors (PRRs) were significantly reduced in expression in cervical mononuclear cells (CMCs) from HESN compared with the new HIV-negative (HIV-N) and HIV-positive (HIV-P) groups. Although baseline levels of secreted cytokines were reduced in CMCs of HESN, they were highly stimulated following exposure to ssRNA40 in vitro. Importantly, cervical epithelial cells from HESN also expressed reduced levels of PRRs, but Toll-like receptor 3 (TLR3) and TLR7 as well as nuclear factor-κB and activator protein 1 were highly expressed and activated. Lastly, inflammatory cytokines interleukin (IL)-1β, IL-8, and RANTES (regulated and normal T cell expressed and secreted) were detected at lower levels in cervicovaginal lavage of HESN compared with the HIV-N and HIV-P groups. Overall, our study reveals a local microenvironment of HIV resistance in the genital mucosa consisting of a finely controlled balance of basal immune quiescence with a focused and potent innate anti-viral response critical to resistance to sexual transmission of HIV-1.

Effect of the Levonorgestrel Intrauterine Device on Genital HIV-1 RNA Shedding Among HIV-1–Infected Women Not Taking Antiretroviral Therapy in Nairobi, Kenya

The effect of the levonorgestrel-releasing intrauterine device (LNG-IUD) on genital HIV-1 RNA shedding and inflammation among 25 HIV-infected women was evaluated. Blood, endocervical, and cervicovaginal lavage samples were collected from HIV-infected women not taking antiretrovirals before LNG-IUD insertion and 1 month, 3 month, and 6 months thereafter. HIV-1 RNA was quantitated by real-time reverse transcriptase-polymerase chain reaction. Inflammatory markers were measured by enzyme immunoassay. Genital HIV-1 RNA shedding and inflammatory markers did not differ between LNG-IUD placement and month 6, with the exception of interleukin 1β that increased (0.42 log10; 95% confidence interval: 0.10 to 0.75). The LNG-IUD did not increase genital HIV-1 RNA shedding after 6 months of use.

Evaluation of a multiplex real time PCR assay for the detection of human papillomavirus infections on self-collected cervicovaginal lavage samples

Self-collection of cervical samples for human papillomavirus (HPV) testing can help to raise the participation rate in cervical cancer screening among non-responders. This study was conducted to compare the analytical and clinical performance of the Abbott RealTime High-Risk HPV Test (RealTime) with the Hybrid Capture 2 assay (HC2) on self-collected cervicovaginal lavage samples. One hundred samples from women referred for colposcopy (reference smears and biopsies in case of abnormalities) were included. Fifty-seven women had a normal cytology, 27 had low grade and 16 had high grade squamous intraepithelial lesions. Fourteen of 49 biopsies (28.6%) were benign, 14 (28.6%) cervical intraepithelial neoplasia 1, 11 (22.4%) cervical intraepithelial neoplasia 2, 8 (16.3%) cervical intraepithelial neoplasia 3 and 2 (4.1%) invasive cancer. The agreement between RealTime and HC2 in the self-collected and in the reference samples was 85% (κ=0.665) and 83% (κ=0.620), respectively. The agreement between self-sampling and the reference smears was higher with RealTime (93%; κ=0.849) than with HC2 (89%; κ=0.741). In the self-collected samples, the sensitivity and specificity for cervical intraepithelial neoplasia 2 or worse of RealTime/HC2 were 81.0%/66.7% (p=0.25) and 53.6%/57.1%, respectively. The sensitivity for cervical intraepithelial neoplasia 3 or worse of RealTime and HC2 were 80.0% and 70.0% (p=1.0). The specificity for cervical intraepithelial neoplasia 3 or worse was 43.6% (RealTime) and 51.3% (HC2). This study shows that RealTime can be used for HR-HPV testing of self-collected lavage samples in a referral population with at least equal quality and performance as HC2.

A second generation cervico-vaginal lavage device shows similar performance as its preceding version with respect to DNA yield and HPV DNA results

BackgroundAttendance rates of cervical screening programs can be increased by offering HPV self-sampling to non-attendees. Acceptability, DNA yield, lavage volumes and choice of hrHPV test can influence effectiveness of the self-sampling procedures and could therefore play a role in recruiting non-attendees. To increase user-friendliness, a frequently used lavage sampler was modified. In this study, we compared this second generation lavage device with the first generation device within similar birth cohorts.MethodsWithin a large self-sampling cohort-study among non-responders of the Dutch cervical screening program, a subset of 2,644 women received a second generation self-sampling lavage device, while 11,977 women, matched for age and ZIP-code, received the first generation model. The second generation device was different in shape, color, lavage volume, and packaging, in comparison to its first generation model. The Cochran’s test was used to compare both devices for hrHPV positivity rate and response rate. To correct for possible heterogeneity between age and ZIP codes in both groups the Breslow-Day test of homogeneity was used. A T-test was utilized to compare DNA yields of the obtained material in both groups.ResultsMedian DNA yields were 90.4 μg/ml (95% CI 83.2-97.5) and 91.1 μg/ml (95% CI 77.8-104.4, p= 0.726) and hrHPV positivity rates were 8.2% and 6.9% (p= 0.419) per sample self-collected by the second - and the first generation of the device (p= 0.726), respectively. In addition, response rates were comparable for the two models (35.4% versus 34.4%, p= 0.654).ConclusionsReplacing the first generation self-sampling device by an ergonomically improved, second generation device resulted in equal DNA yields, comparable hrHPV positivity rates and similar response rates. Therefore, it can be concluded that the clinical performance of the first and second generation models are similar. Moreover, participation of non-attendees in cervical cancer screening is probably not predominantly determined by the type of self-collection device.

Antiviral Activity of Trappin-2 and Elafin In Vitro and In Vivo against Genital Herpes

Serine protease inhibitor elafin (E) and its precursor, trappin-2 (Tr), have been associated with mucosal resistance to HIV-1 infection. We recently showed that Tr/E are among principal anti-HIV-1 molecules in cervicovaginal lavage (CVL) fluid, that E is ∼130 times more potent than Tr against HIV-1, and that Tr/E inhibited HIV-1 attachment and transcytosis across human genital epithelial cells (ECs). Since herpes simplex virus 2 (HSV-2) is a major sexually transmitted infection and risk factor for HIV-1 infection and transmission, we assessed Tr/E contribution to defense against HSV-2. Our in vitro studies demonstrated that pretreatment of endometrial (HEC-1A) and endocervical (End1/E6E7) ECs with human Tr-expressing adenovirus (Ad/Tr) or recombinant Tr/E proteins before or after HSV-2 infection resulted in significantly reduced virus titers compared to those of controls. Interestingly, E was ∼7 times more potent against HSV-2 infection than Tr. Conversely, knockdown of endogenous Tr/E by small interfering RNA (siRNA) significantly increased HSV-2 replication in genital ECs. Recombinant Tr and E reduced viral attachment to genital ECs by acting indirectly on cells. Further, lower viral replication was associated with reduced secretion of proinflammatory interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) and decreased NF-κB nuclear translocation. Additionally, protected Ad/Tr-treated ECs demonstrated enhanced interferon regulatory factor 3 (IRF3) nuclear translocation and increased antiviral IFN-β in response to HSV-2. Lastly, in vivo studies of intravaginal HSV-2 infection in Tr-transgenic mice (Etg) showed that despite similar virus replication in the genital tract, Etg mice had reduced viral load and TNF-α in the central nervous system compared to controls. Collectively, this is the first experimental evidence highlighting anti-HSV-2 activity of Tr/E in female genital mucosa.

IL-22 levels are associated with Trichomonas vaginalis infection in the lower genital tract.

IL-22 has important functions at mucosal surfaces, including the induction of antimicrobial peptides and maintenance of epithelium. However, IL-22 has not been investigated in the genital tract during TV infection. Women who visited an STD clinic and women from a cohort with frequent Trichomoniasis were studied. IL-22, IL-17, and antimicrobial peptides were measured in cervicovaginal lavage by ELISA. In women visiting the STD clinic, those without STDs (n = 10) had a median IL-22 of 0 pg/mL, while women with infections (n = 30) had 27 pg/mL (P = 0.04). In the cohort, women with Trichomoniasis (n = 19) had significantly higher IL-22 than women with no infections (n = 21, 74 versus 0 pg/mL, P = 0.0001). IL-17 was also significantly increased in Trichomoniasis, and there was a correlation between IL-22 and IL-17 (P = 0.001). IL-22 is increased in STDs generally and in Trichomoniasis specifically suggesting an antimicrobial response of the mucosa and an epithelial repair process induced by the STDs.