High Risk HPV Contamination of Endocavity Vaginal Ultrasound Probes: An Underestimated Route of Nosocomial Infection?

Background

Endocavity ultrasound is seen as a harmless procedure and has become a common gynaecological procedure. However without correct disinfection, it may result in nosocomial transmission of genito-urinary pathogens, such as high-risk Human Papillomavirus (HR-HPV). We aimed to evaluate the currently recommended disinfection procedure for covered endocavity ultrasound probes, which consists of “Low Level Disinfection” (LLD) with “quaternary ammonium compounds” containing wipes.

Methods

From May to October 2011 swabs were taken from endovaginal ultrasound probes at the Gynecology Department of the Lyon University Hospital. During the first phase (May–June 2011) samples were taken after the ultrasound examination and after the LLD procedure. In a second phase (July–October 2011) swab samples were collected just before the probe was used. All samples were tested for the presence of human DNA (as a marker for a possible transmission of infectious pathogens from the genital tract) and HPV DNA with the Genomica DNA microarray (35 different HPV genotypes).

Results

We collected 217 samples before and 200 samples after the ultrasound examination. The PCR was inhibited in two cases. Human DNA was detected in 36 (18%) post-examination samples and 61 (28%) pre-examination samples. After the ultrasound LLD procedure, 6 (3.0%) samples contained HR-HPV types (16, 31, 2×53 and 58). Similarly, HPV was detected in 6 pre-examination samples (2.7%). Amongst these 4 (1.9%) contained HR-HPV (types 53 and 70).

Conclusion

Our study reveals that a considerable number of ultrasound probes are contaminated with human and HR-HPV DNA, despite LLD disinfection and probe cover. In all hospitals, where LLD is performed, the endovaginal ultrasound procedure must therefore be considered a source for nosocomial HR-HPV infections. We recommend the stringent use of high-level disinfectants, such as glutaraldehyde or hydrogen peroxide solutions.     

The Vaginal Microbiota: What Have We Learned after a Decade of Molecular Characterization?

We conducted a systematic review of the Medline database (U.S. National Library of Medicine, National Institutes of Health, Bethesda, MD, U.S.A) to determine if consistent molecular vaginal microbiota (VMB) composition patterns can be discerned after a decade of molecular testing, and to evaluate demographic, behavioral and clinical determinants of VMB compositions. Studies were eligible when published between 1 January 2008 and 15 November 2013, and if at least one molecular technique (sequencing, PCR, DNA fingerprinting, or DNA hybridization) was used to characterize the VMB. Sixty three eligible studies were identified. These studies have now conclusively shown that lactobacilli-dominated VMB are associated with a healthy vaginal micro-environment and that bacterial vaginosis (BV) is best described as a polybacterial dysbiosis. The extent of dysbiosis correlates well with Nugent score and vaginal pH but not with the other Amsel criteria. Lactobacillus crispatus is more beneficial than L. iners. Longitudinal studies have shown that a L. crispatus-dominated VMB is more likely to shift to a L. iners-dominated or mixed lactobacilli VMB than to full dysbiosis. Data on VMB determinants are scarce and inconsistent, but dysbiosis is consistently associated with HIV, human papillomavirus (HPV), and Trichomonas vaginalis infection. In contrast, vaginal colonization with Candida spp. is more common in women with a lactobacilli-dominated VMB than in women with dysbiosis. Cervicovaginal mucosal immune responses to molecular VMB compositions have not yet been properly characterized. Molecular techniques have now become more affordable, and we make a case for incorporating them into larger epidemiological studies to address knowledge gaps in etiology and pathogenesis of dysbiosis, associations of different dysbiotic states with clinical outcomes, and to evaluate interventions aimed at restoring and maintaining a lactobacilli-dominated VMB.     

Composition of the Vaginal Microbiota in Women of Reproductive Age – Sensitive and Specific Molecular Diagnosis of Bacterial Vaginosis Is Possible?

Background and Objective

Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis.

Materials and Methods

Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3–V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated.

Results

Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor.

Conclusions

Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.     

Development and Evaluation of an In Vitro Vaginal Model for Assessment of Drug’s Biopharmaceutical Properties: Curcumin

Vaginal administration is a promising alternative to the per-oral route in achieving systemic or local therapeutic effects, when intestinal drug absorption is hindered by problematic biopharmaceutical drug properties. The aim of this study was to establish an in vitro vaginal model and use it to characterize biopharmaceutical properties of liposomally associated curcumin destined for vaginal delivery. The in vitro permeability, metabolism, and tissue retention of high/low permeable compounds were assessed on cow vaginal mucosa and compared to the permeabilities determined through Caco-2 cells and rat jejunum in vitro. The results showed that the intestinal mucosa was superior to the vaginal one in categorizing drugs based on their permeabilities in high/low permeable classes. Passive diffusion was found to be the main mechanism of drug penetration through vaginal mucosa and it was not affected by transporter–enzyme alliance, as their expression/activity was significantly reduced compared to the intestinal tract. Curcumin permeability from the solution form was the lowest of all tested substances due to its significant tissue retention and curcumin–mucus interactions. The permeability of liposomally associated curcumin was even lower but the binding of liposomally associated curcumin to the vaginal tissue was significantly higher. The permeability and tissue retention of liposomal curcumin were vesicle size dependent. Vaginal application of liposomally associated curcumin provides relatively high levels of curcumin in vaginal tissue, with limited systemic absorption.KEY WORDS: curcumin, intestinal models, liposomes, permeability, vaginal mucosa     

How Accurate Is Preoperative Evaluation of Pelvic Organ Prolapse in Women Undergoing Vaginal Reconstruction Surgery?

Objective

To evaluate the differences between the in-office and intraoperative techniques used to evaluate pelvic organ prolapse.

Materials and Methods

A prospective study included 25 women undergoing vaginal reconstruction surgery including vaginal hysterectomy for pelvic organ prolapse. The outpatient pelvic and site-specific vaginal examination was performed in the lithotomy position with the Valsalva maneuver. Repeated intraoperative examination was performed under general anesthesia with standard mild cervical traction. The Pelvic Organ Prolapse Quantification system (POPQ) was used for both measurements and staging. The values found under the two conditions were compared.

Results

The intraoperative POPQ-measurements values were significantly higher than the outpatient values for apical wall prolapse in 17/25 (68%) women and for anterior wall prolapse in 8/25 (32%) women. There was not a significant difference in the posterior wall where increase in staging was shown in 3/25 (12%) patients.

Conclusions

Clinicians and patients should be alert to the possibility that pelvic organ measurements performed under general anesthesia with mild traction may be different from preoperative evaluation.     

The Role of 17β-Estrogen in Candida albicans Adhesion on Human Vaginal Epithelial Cells via FAK Phosphorylation

Purposes

To investigate the role of 17β-estrogen in Candida albicans (C. albicans) adhesion on human vaginal epithelial cells in vulvovaginal candidiasis (VVC).

Methods

The vaginal epithelial cell line, VK2/E6E7, was used to study the estrogen-induced molecular events between C. albicans and cells. An adhesion study was performed to evaluate the involvement of the estrogen-dependent focal adhesion kinase (FAK) activation in cell adhesion. The phosphorylation status of FAK and estrogen receptor α (ERα) upon estrogen challenge was assessed by western blotting. Specific inhibitors for ERα were used to validate the involvement of ERα–FAK signaling cascade.

Results

A transient activation of ERα and FAK was observed following the stimulation with 1000 nM estrogen for 48 h, as well as the increased average number of C. albicans adhering to each vaginal epithelial cell. Estrogen-induced activation of ERa and FAK was inhibited by the specific inhibitor of ERα, especially when the inhibitor reached a 10 μM concentration and allowed to act for 12 h. Simultaneously, a decrease in the number of adherent C. albicans was observed. However, this inhibitory effect diminished as the concentration of estrogen increased.

Conclusion

FAK and ERα signaling cascades were involved in the early interaction between the vaginal epithelial cells and C. albicans, which appeared to be linked with the enhanced cell adhesion leading to VVC and promoted by a certain concentration of estrogen.

     

Location and Dynamics of the Immunodominant CD8 T Cell Response to SIVΔnef Immunization and SIVmac251 Vaginal Challenge

Live-attenuated SIV vaccines (LAVs) have been the most effective to date in preventing or partially controlling infection by wild-type SIV in non-human primate models of HIV-1 transmission to women acting by mechanisms of protection that are not well understood. To gain insights into mechanisms of protection by LAVs that could aid development of effective vaccines to prevent HIV-1 transmission to women, we used in situ tetramer staining to determine whether increased densities or changes in the local distribution of SIV-specific CD8 T cells correlated with the maturation of SIVΔnef vaccine-induced protection prior to and after intra-vaginal challenge with wild-type SIVmac251. We evaluated the immunodominant Mamu-A1*001:01/Gag (CM9) and Mamu-A1*001:01/Tat (SL8) epitope response in genital and lymphoid tissues, and found that tetramer+ cells were present at all time points examined. In the cervical vaginal tissues, most tetramer+ cells were distributed diffusely throughout the lamina propria or co-localized with other CD8 T cells within lymphoid aggregates. The distribution and densities of the tetramer+ cells at the portal of entry did not correlate with the maturation of protection or change after challenge. Given these findings, we discuss the possibility that changes in other aspects of the immune system, including the quality of the resident population of virus-specific effector CD8 T cells could contribute to maturation of protection, as well as the potential for vaccine strategies that further increase the size and quality of this effector population to prevent HIV-1 transmission.     

HSV-2-Driven Increase in the Expression of α4β7 Correlates with Increased Susceptibility to Vaginal SHIVSF162P3 Infection

The availability of highly susceptible HIV target cells that can rapidly reach the mucosal lymphoid tissues may increase the chances of an otherwise rare transmission event to occur. Expression of α₄β₇ is required for trafficking of immune cells to gut inductive sites where HIV can expand and it is expressed at high level on cells particularly susceptible to HIV infection. We hypothesized that HSV-2 modulates the expression of α₄β₇ and other homing receptors in the vaginal tissue and that this correlates with the increased risk of HIV acquisition in HSV-2 positive individuals. To test this hypothesis we used an in vivo rhesus macaque (RM) model of HSV-2 vaginal infection and a new ex vivo model of macaque vaginal explants. In vivo we found that HSV-2 latently infected RMs appeared to be more susceptible to vaginal SHIV_SF162P3 infection, had higher frequency of α₄β₇ ^high CD4⁺ T cells in the vaginal tissue and higher expression of α₄β₇ and CD11c on vaginal DCs. Similarly, ex vivo HSV-2 infection increased the susceptibility of the vaginal tissue to SHIV_SF162P3. HSV-2 infection increased the frequencies of α₄β₇ ^high CD4⁺ T cells and this directly correlated with HSV-2 replication. A higher amount of inflammatory cytokines in vaginal fluids of the HSV-2 infected animals was similar to those found in the supernatants of the infected explants. Remarkably, the HSV-2-driven increase in the frequency of α₄β₇ ^high CD4⁺ T cells directly correlated with SHIV replication in the HSV-2 infected tissues. Our results suggest that the HSV-2-driven increase in availability of CD4⁺ T cells and DCs that express high levels of α₄β₇ is associated with the increase in susceptibility to SHIV due to HSV-2. This may persists in absence of HSV-2 shedding. Hence, higher availability of α₄β₇ positive HIV target cells in the vaginal tissue may constitute a risk factor for HIV transmission.     

Vaginal delivery and continuous epidural analgesia: should we change our clinical approach?

The aim of the study was to investigate the effects of continuous epidural analgesia (EA) on the course of vaginal delivery with an emphasis on duration of labor and instrumental interventions. In a prospective 2-year trial, the study group included singleton vaginal births between 35 and 41 gestational weeks with a vertex fetus, in which continuous EA with bupivacaine or chirocaine in concentration of 0.125% combined with 2-4 microg of fentanyl or 0.5 microg of sufenta was used. The control group was created randomly from laboring patients with singleton pregnancies but without EA. The groups were adjusted for epidemiological characteristics and compared regarding the obstetric data and perinatal outcome. Student t-test and Mann-Whitney U-test were performed for normally and non-normally distributed results, respectively. Out of 1284 patients, 551 pregnant women were included in the study group and 733 in the control group. The statistically significant differences between the groups related to duration of the first and second stage of labor, frequency of premature rupture of membranes, intrapartal complications, and incidence of operative deliveries were found. Both stages of labor were significantly protracted and the incidence of operative deliveries was higher in the study group of patients compared with controls. There is a need for an active obstetric approach and management of vaginal deliveries of women who receive continuous EA, particularly if it is medically indicated.     

Newly Isolated Lactobacilli strains from Algerian Human Vaginal Microbiota: Lactobacillus fermentum Strains Relevant Probiotic’s Candidates

Probiotics and Antimicrobial Proteins - Lactobacilli strains are considered as a preventive means for treatment of vaginal infections or post-antibiotic treatment to repopulate the vaginal mucosa....     

SPL7013 Gel (VivaGel?) Retains Potent HIV-1 and HSV-2 Inhibitory Activity following Vaginal Administration in Humans

SPL7013 Gel (VivaGel^®) is a microbicide in development for prevention of HIV and HSV. This clinical study assessed retention and duration of antiviral activity following vaginal administration of 3% SPL7013 Gel in healthy women. Participants received 5 single doses of product with ≥5 days between doses. A cervicovaginal fluid (CVF) sample was collected using a SoftCup™ pre-dose, and immediately, or 1, 3, 12 or 24 h post-dose. HIV-1 and HSV-2 antiviral activities of CVF samples were determined in cell culture assays. Antiviral activity in the presence of seminal plasma was also tested. Mass and concentration of SPL7013 in CVF samples was determined. Safety was assessed by reporting of adverse events. Statistical analysis was performed using the Wilcoxon signed-rank test with Bonferroni adjustment; p≤0.003 was significant. Eleven participants completed the study. Inhibition of HIV-1 and HSV-2 by pre-dose CVF samples was negligible. CVF samples obtained immediately after dosing almost completely inhibited (median, interquartile range) HIV-1 [96% (95,97)] and HSV-2 [86% (85,94)], and activity was maintained in all women at 3 h (HIV-1 [96% (95,98), p = 0.9]; HSV-2 [94% (91,97), p = 0.005]). At 24 h, >90% of initial HIV-1 and HSV-2 inhibition was maintained in 6/11 women. SPL7013 was recovered in CVF samples obtained at baseline (46% of 105 mg dose). At 3 and 24 h, 22 mg and 4 mg SPL7013, respectively, were recovered. More than 70% inhibition of HIV-1 and HSV-2 was observed if there was >0.5 mg SPL7013 in CVF samples. High levels of antiviral activity were retained in the presence of seminal plasma. VivaGel was well tolerated with no signs or symptoms of vaginal, vulvar or cervical irritation reported. Potent antiviral activity was observed against HIV-1 and HSV-2 immediately following vaginal administration of VivaGel, with activity maintained for at least 3 h post-dose. The data provide evidence of antiviral activity in a clinical setting, and suggest VivaGel could be administered up to 3 h before coitus.

Trial Registration

The study is registered at ClinicalTrials.gov under identifier: {"type":"clinical-trial","attrs":{"text":"NCT00740584","term_id":"NCT00740584"}}NCT00740584     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Selection of optimal variants of Gō-like models of proteins through studies of stretching

The Gō-like models of proteins are constructed based on the knowledge of the native conformation. However, there are many possible choices of a Hamiltonian for which the ground state coincides with the native state. Here, we propose to use experimental data on protein stretching to determine what choices are most adequate physically. This criterion is motivated by the fact that stretching processes usually start with the native structure, in the vicinity of which the Gō-like models should work the best. Our selection procedure is applied to 62 different versions of the Gō model and is based on 28 proteins. We consider different potentials, contact maps, local stiffness energies, and energy scales—uniform and nonuniform. In the latter case, the strength of the nonuniformity was governed either by specificity or by properties related to positioning of the side groups. Among them is the simplest variant: uniform couplings with no i, i + 2 contacts. This choice also leads to good folding properties in most cases. We elucidate relationship between the local stiffness described by a potential which involves local chirality and the one which involves dihedral and bond angles. The latter stiffness improves folding but there is little difference between them when it comes to stretching.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Association of the subunits of the calcium-independent receptor of α-latrotoxin

CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.