RAFT-Derived Polymethacrylates as a Superior Treatment for Recurrent Vulvovaginal Candidiasis by Targeting Biotic Biofilms and Persister Cells.
ABSTRACT: Background:Vulvovaginal candidiasis (VVC) is a common infection in need of more effective treatment. Formation of epithelium-associated Candida biofilms and the presence of persister cells are among the major contributing factors to the recurrence of this condition. We have previously developed RAFT-derived polymethacrylates that are effective in killing C. albicans biofilms in vitro. This study aimed to examine the clinical potential of polymethacrylates as antifungals for treatment of recurrent VVC (RVVC). Methods:A mouse model of VVC was used to establish vaginal epithelium-associated biofilms, using C. albicans isolates from VVC/RVVC patients. A comparison was made of the efficacies of polymethacrylates and conventional antifungals, clotrimazole and nystatin, in killing Candida in epithelium-associated biofilms in vivo. Ex vivo biofilms were used for Candida population profiling and to quantify persister cells in vaginal epithelia. The potency of polymethacrylates and conventional antifungals against persister cells, either as sole agents or in combination, was assessed. Results:Polymethacrylates showed negligible local toxicity, resistance to vaginal acidity, and outstanding in vivo activity against vaginal epithelium-associated C. albicans biofilms. In vivo tests polymethacrylates outperformed the conventional antifungals, nystatin and clotrimazole at concentrations 50 times below the over-the-counter concentrations. Using polymethacrylates was associated with fewer persister cells, and better eradication of persister cells pre-selected by conventional antifungals. Conclusion:This study systematically assessed the clinical potential of RAFT-derived polymethacrylates as an effective treatment for VVC/RVVC in a mouse model. Polymethacrylates effectively killed vaginal epithelium-related C. albicans in vivo by specially targeting biotic biofilms and persister cells. Treatment presented negligible local toxicity.
Project description:Purpose:Antifungal resistance and virulence properties of Candida albicans (C. albicans) are growing health problems worldwide. The present study aims to investigate the effect of Zinc Oxide (ZnO) nanoparticles and Nystatin on SAP1-3 genes expression in C. albicans isolates of females with Vulvovaginal Candidiasis (VVC) isolated from Sayad Shirazi Obstetrics and Gynecology Hospital in Northeastern Iran during 2017-2018. Patients and Methods:In this descriptive-analytic study, vaginal samples were collected from 280 VVC women. 196 (70%) of C. albicans isolates were identified by phenotypic and ITS genotypic methods. Susceptibility to Fluconazole C. albicans isolates was determined by the disk diffusion method. Detection of ERG11 gene was done by RT-PCR technique. Results:It was revealed that PCR amplified the ERG11 gene in all of the Fluconazole-resistant isolates. Real-time PCR was used to survey the effects of 3±1.7µg/mL concentrations of ZnO nanoparticles and Nystatin on expression of SAP1-3 genes before and after treatment. 186 (95%) susceptible C. albicans and 10 (5%) Fluconazole-resistant C. albicans isolates from VVC were exposed to sub-minimum inhibitory concentrations (Sub-MIC) of ZnO-np (range=0.02-12 ?g/mL). Sub-MIC concentration was used for each strain, which reduced the expression of SAP1-3 genes to 1.8 MIC in the vaginal swabs. The observed reduction in gene expression was significant for both ZnO nanoparticles and Nystatin (P=0.01 and P=0.07, respectively). Conclusion:ZnO as antifungal agent can well reduce the growth and gene expression of SAP1-3 in the pathogenesis of VVC.
Project description:BACKGROUND:Vulvovaginal candidiasis (VVC) is a common infection affecting women worldwide. Reports of patterns/risk factors/trends for episodic/recurrent VVC (RVVC) are largely outdated. The purpose of this study was to obtain current patient perspectives of several aspects of VVC/RVVC. METHODS:Business cards containing on-line survey information were distributed to healthy volunteers and patients seeking standard, elective, or referral gynecologic care in university-affiliated Obstetrics/Gynecology clinics. The internet-based questionnaire was completed by 284 non-pregnant women (78% Caucasian, 14% African American, 8% Asian). RESULTS:The majority of the participants (78%) indicated a history of VVC with 34% defined as having RVVC. The most common signs/symptoms experienced were itching, burning and redness with similar ranking of symptoms among VVC and RVVC patients. Among risk factors, antibiotic use ranked highest followed by intercourse, humid weather and use of feminine hygiene products. A high number of respondents noted 'no known cause' (idiopathic episodes) that was surprisingly similar among women with a history of either VVC or RVVC. VVC/RVVC episodes reported were primarily physician-diagnosed (73%) with the remainder mostly reporting self-diagnosis and treating with over-the-counter (OTC) medications. Most physician-diagnosed attacks utilized a combination of pelvic examination and laboratory tests followed by prescribed antifungals. Physician-treated cases achieved a higher level of symptom relief (84%) compared to those who self-medicated (57%). The majority of women with RVVC (71%) required continual or long-term antifungal medication as maintenance therapy to control symptoms. CONCLUSIONS:Current patient perspectives closely reflect historically documented estimates of VVC/RVVC prevalence and trends regarding symptomatology, disease management and post-treatment outcomes.
Project description:The ability to tolerate Candida albicans, a human commensal of the gastrointestinal tract and vagina, implicates that host defense mechanisms of resistance and tolerance cooperate to limit fungal burden and inflammation at the different body sites. We evaluated resistance and tolerance to the fungus in experimental and human vulvovaginal candidiasis (VVC) as well as in recurrent VVC (RVVC). Resistance and tolerance mechanisms were both activated in murine VVC, involving IL-22 and IL-10-producing regulatory T cells, respectively, with a major contribution by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 was responsible for the production of tolerogenic kynurenines, such that replacement therapy with kynurenines restored immunoprotection to VVC. In humans, two functional genetic variants in IL22 and IDO1 genes were found to be associated with heightened resistance to RVVC, and they correlated with increased local expression of IL-22, IDO1 and kynurenines. Thus, IL-22 and IDO1 are crucial in balancing resistance with tolerance to Candida, their deficiencies are risk factors for RVVC, and targeting tolerance via therapeutic kynurenines may benefit patients with RVVC.
Project description:The purpose of this study was to prospectively evaluate the impact of the use of L. plantarum I1001 applied vaginally on Vulvovaginal Candidiasis (VVC) time-until-recurrence after treatment with single-dose vaginal clotrimazole. This was a clinical open-label, prospective study of two non-randomized parallel cohorts with symptomatic acute VVC: (1) 33 sexually active women 18-50 years old, prescribed a standard single-dose 500 mg vaginal tablet of clotrimazole followed by vaginal tablets with L. plantarum I1001 as adjuvant therapy, and (2) 22 women of similar characteristics but prescribed single-dose clotrimazole only. Use of the probiotic and factors that might influence recurrence risk (age, recurrent VVC within previous year, antibiotic prior to study enrolment, diaphragm or IUD contraception, among others) were included in a multivariate Cox regression model to adjust for potential between-cohort differences. Probiotic use was associated with a three-fold reduction in the adjusted risk of recurrence (HR [95 %CI]: 0.30 [0.10-0.91]; P?=?0.033). Adjusted free-survival recurrence was 72.83 % and 34.88 % for the probiotic and control groups, respectively. A higher cumulative recurrence was also observed in cases with use of antibiotics prior to enrolment (HR [95 %CI]: 10.46 [2.18-50.12]; P?=?0.003). Similar findings were found at six months after azole treatment in women with RVVC. Overall, good compliance with the probiotic was reported for 91.3 % of women. The study suggests that follow-up therapy with vaginal tablets with L. plantarum I1001 could increase the effectiveness of single-dose 500 mg clotrimazole at preventing recurrence of VVC, an effect that was also observed in women with recurrent vulvovaginal candidiasis (RVVC) after six months of azole treatment.
Project description:In acute vulvovaginal candidiasis (VVC), the fungus Candida albicans activates inflammasome receptors of vaginal epithelial cells through the production of virulence and immuno-inflammatory factors. Here, we show that in VVC patients, genes encoding some of the above factors (SAP2, SAP5, SAP6, ECE1, and HWP1) are expressed in a correlated fashion. Cytological observations pointed out that pseudohyphal filaments with yeast cells are dominant at the acidic vaginal pH, and this is coupled with co-expression, at roughly similar level, of SAP2, a typical yeast and ECE1, a typical hyphae-associated genes. In contrast, vigorous hyphal growth dominated at the neutral vaginal pH of mice experimentally infected with C. albicans isolates from VVC subjects, and this is coupled with a high ratio of ECE1 to SAP2 expression. We suggest that the pseudohyphal rather than true hyphal cells of C. albicans play a critical role in VVC, possibly through the activity of multiple inflammasome inducers.
Project description:OBJECTIVE:Vulvovaginal candidiasis (VVC) is a common vaginal infection. Risk factors include diabetes, antibiotic use and pregnancy. Candida albicans is the most common species identified but non-C. albicans species appear to be more commonly associated with VVC in some Asian and African countries. We had studied the distribution of Candida species in Spanish and immigrants' women residents in Spain. METHODS:Retrospective study of vaginal yeast cultures between 2015 and 2018. RESULTS:A total of 2,283 vaginal yeast cultures were collected. Candida spp. was detected in 25.7% from Spanish women and in 28.5% from immigrants (no significant differences). Immigrants have higher rates of vaginal candidiasis compared other studies in Spain. C. albicans was the most common species isolated (82.4%). CONCLUSIONS:There were no differences in vaginal candidiasis rate between Spanish and immigrants' women. Immigrants consulted proportionally more compared with the Spanish women.
Project description:Vulvovaginal candidiasis (VVC) is a common fungal infection caused by Candida albicans. The antifungal therapy represents the standard of care but due to the high costs of treatment and to the inability to prevent recurrences, the development of alternative therapeutic approaches is much-awaited. Recently, we have shown that the pathogenesis of C. albicans in the gut is modulated by IL-9, a pleiotropic cytokine able to promote both inflammation and tolerance during C. albicans infection. Herein, by using a mouse model of VVC, we similarly demonstrated that IL-9 might exert a dual role in VVC by contributing to inflammation during the initial immune activation and promoting resolution thereafter. Specifically, IL-9 has a pro-inflammatory activity at the onset of VVC by promoting NLRP3 inflammasome activity and mucosal mast cells expansion but a tolerogenic role in the resolution phase by promoting IL-1Ra production and connective tissue mast cells activation. We further show that a timely IL-9 neutralization at the onset of the inflammatory response ameliorated symptoms and vaginal pathology. Given that vaginal fluids from patients with recurrent VVC had higher levels of IL-9, these findings, by providing novel insights into the pathogenesis of VVC, may pave the way for alternative therapeutic strategies based on IL-9 neutralization.
Project description:Vulvovaginal candidiasis (VVC) is a common mucosal infection caused by Candida spp., most frequently by Candida albicans, which may become recurrent and severely impacting the quality of life of susceptible women. Although it is increasingly being recognized that mucosal damage is mediated by an exaggerated inflammatory response, current therapeutic approaches are only based on antifungals that may relieve the symptomatology, but fail to definitely prevent recurrences. The unrestrained activation of the NLRP3 inflammasome with continuous production of IL-1? and recruitment of neutrophils is recognized as a pathogenic factor in VVC. We have previously shown that IL-22 is required to dampen pathogenic inflammasome activation in VVC via the NLRC4/IL-1Ra axis. However, IL-22 also regulates IL-18, a product of the inflammasome activity that regulates IL-22 expression. Here we describe a cross-regulatory circuit between IL-18 and IL-22 in murine VVC that is therapeutically druggable. We found that IL-18 production was dependent on IL-22 and NLRC4, and that IL-18, in turn, contributes to IL-22 activity. Like in IL-22 deficiency, IL-18 deficiency was associated with an increased susceptibility to VVC and unbalanced Th17/Treg response, suggesting that IL-18 can regulate both the innate and the adaptive responses to the fungus. Administration of the microbial metabolite indole-3-aldehyde, known to stimulate the production of IL-22 via the aryl hydrocarbon receptor (AhR), promoted IL-18 expression and protection against Candida infection. Should low levels of IL-18 be demonstrated in the vaginal fluids of women with recurrent VVC, targeting the AhR/IL-22/IL-18 pathway could be exploited for future therapeutic approaches in VVC. This study suggests that a deeper understanding of the mechanisms regulating inflammasome activity may lead to the identification of novel targets for intervention in VVC.
Project description:Candida albicans is a major fungal pathogen causing lethal infections in immunocompromised patients. C. albicans forms antifungal tolerant biofilms contributing significantly to therapeutic failure. The recently established haploid C. albicans biofilm model provides a new toolbox to uncover the mechanism governing the higher antifungal tolerance of biofilms. Here, we comprehensively examined the proteomics and antifungal susceptibility of standard diploid (SC5314 and BWP17) and stable haploid (GZY792 and GZY803) strains of C. albicans biofilms. Subsequent downstream analyses identified alkyl hydroperoxide reductase 1 (AHP1) as a critical determinant of C. albicans biofilm's tolerance of amphotericin B. At 32 ?g/ml of amphotericin B, GZY803 haploid biofilms showed 0.1% of persister population as compared with 1% of the diploid biofilms. AHP1 expression was found to be lower in GZY803 biofilms, and AHP1 overexpression in GZY803 restored the percentage of persister population. Consistently, deleting AHP1 in the diploid strain BWP17 caused a similar increase in amphotericin B susceptibility. AHP1 expression was also positively correlated with the antioxidant potential. Furthermore, C. albicans ira2?/? biofilms were susceptible to amphotericin B and had a diminished antioxidant capacity. Interestingly, AHP1 overexpression in the ira2?/? strain restored the antioxidant potential and enhanced the persister population against amphotericin B, and shutting down the AHP1 expression in ira2?/? biofilms reversed the effect. In conclusion, we provide evidence that the AHP1 gene critically determines the amphotericin B tolerance of C. albicans biofilms possibly by maintaining the persisters' antioxidant capacity. This finding will open up new avenues for developing therapies targeting the persister population of C. albicans biofilms. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD004274.
Project description:A Phase 1b/2a clinical trial of NDV-3A vaccine containing a Candida albicans recombinant Als3 protein formulated with alum protected women <40?years old from recurrent vulvovaginal candidiasis (RVVC). We investigated the potential use of anti-Als3p sera as surrogate marker of NDV-3A efficacy. Pre- and post-vaccination sera from subjects who experienced recurrence of vulvovaginal candidiasis (R) vs. those who were recurrence-free [non-recurrent (NR)] were evaluated. Anti-Als3p antisera obtained were evaluated for (1) titer and subclass profile and (2) their ability to influence C. albicans virulence traits including hyphal elongation, adherence to plastic, invasion of vaginal epithelial cells, biofilm formation on plastic and catheter material, and susceptibility to neutrophil killing in vitro. Serum IgG titers in NR patients were consistently higher than in R patients, particularly for anti-Als3 subclass IgG2. Sera from vaccinated NR patients reduced hyphal elongation, adhesion to plastic, invasion of vaginal epithelial cells, and biofilm formation significantly more than pre-immune sera, or sera from R- or placebo-group subjects. Pre-adsorption of sera with C. albicans germ tubes eliminated these effects, while heat inactivation did not. Finally, sera from NR subjects enhanced neutrophil-mediated killing of C. albicans relative to pre-immune sera or sera from R patients. Our results suggest that higher Als3p antibody titers are associated with protection from RVVC, attenuate C. albicans virulence, and augment immune clearance of the fungus in vitro. Thus, Als3p serum IgG antibodies are likely useful markers of efficacy in RVVC patients vaccinated with NDV-3A.