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Urine is an organic fluid produced by the kidney, and its analysis is one of the most requested laboratory tests by clinicians. The ionic composition of urine has been shown to be a good health indicator: it is useful for the diagnosis of several diseases, as well as monitoring therapeutics. This review considers laboratorial techniques that have been used throughout time for the quantification of ions in urine, and also considers some methodologies that can potentially be used in clinical laboratories for this kind of analysis. Those methods include gravimetry, titration, flame emission spectrophotometry (flame photometry), fluorimetry, potentiometry (ion selective electrodes), ion chromatography, electrophoresis, kinetic colorimetric tests, enzymatic colorimetric tests, flow cytometry, atomic absorption, plasma atomic emission spectrometry, and paper-based devices. Sodium, potassium, chloride, calcium, and magnesium are among the most important physiological ions, and their determination is frequently requested in hospitals. There have been many advances regarding the analysis of these ions in 24 h urine. However, there is still some way to go concerning the importance of intracellular ions in this type of sample as well as the use of occasional urine for monitoring these parameters.

Human papillomavirus (HPV)-associated cervical cancer is the most common cancer among women worldwide. The treatment options are strongly related to increased infertility in women. Imiquimod (IQ) is an imidazoquinoline, which has proven antiviral effects against persistent HPV infection by activating immune cells via Toll-like receptors 7/8 when formulated in carriers, like nanogels, for topical use. An effective alternative to conventional therapies is the nanoparticle drug delivery system. We studied lipidic nanoparticles with IQ (Lipo IQ) and functionalized them with a DNA aptamer, AT11 (Lipo IQ AT11), to improve the selectivity for cervical cancer cells combined with the efficacy of essential oils. The formulations showed that the physicochemical properties are adequate for vaginal drug delivery and have antimicrobial activity at higher concentrations (with MIC 50 starting from 0.625%). The final formulations exhibited cytotoxicity in cancer cells, enhanced by essential oils without affecting healthy cells, resulting in less than 10% cell viability in HeLa cells and over 60% in NHDF cells. Essential oils potentiate Lipo IQ's effectiveness, while AT11 increases the selectivity for cervical cancer cells. As suggested by the results of the permeation assay, the formulations were internalized by the cancer cells. Overall, the obtained results suggested that the synergistic effect of the essential oils and the nanosystem potentiate the cytotoxic effect of Lipo IQ and that Lipo IQ AT11 promotes selectivity towards cancer cells.

Humulus lupulus extracts have in their composition different molecules, such as polyphenols, α-acids, β-acids, and hydrocarbons, which contribute to the plant's medicinal properties. These molecules are associated with antimicrobial, antioxidant and anti-inflammatory activities., Objective: This work focuses on the evaluation of H. lupulus biological activities, with the aim of evaluating its potential for inclusion in cosmetic formulations., Methods: Two distinct aqueous extracts and two hydrolates obtained via hydrodistillation were evaluated. These include the flower parts (FE, FH) and the mix of aboveground parts (ME, MH). The chemical profiles for both aqueous extracts and hydrolates were identified by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Antimicrobial, antioxidant, cytotoxicity, and anti-inflammatory activity were tested in vitro using standard methods., Results: Rutin was the major compound found in FE (40.041 μg mg -1 of extract) and ME (2.909 μg mg -1 of extract), while humulenol II was the most abundant compound in hydrolates (FH: 20.83%; MH: 46.80%). Furthermore, FE was able to inhibit the growth of Staphylococcus aureus and Staphylococcus epidermis with MIC values of 50% and 25% (v/v), respectively. FH showed the same effect in Staphylococcus aureus (50% v/v). FH evidenced poor antioxidant potential in DPPH scavenging test and demonstrated significant antioxidant and anti-inflammatory effects by reducing (***p < 0.001) intracellular reactive oxygen species (ROS), NO (nitric oxide) levels (***p < 0.001) and cyclooxygenase-2 (COX-2) protein expression (***p < 0.001) in lipopolysaccharide (LPS)-stimulated macrophages. Nevertheless, it is important to note that FH exhibited cytotoxicity at high concentrations in 3T3 fibroblasts and RAW 264.7 macrophages., Conclusion: The studied H. lupulus aqueous extracts and hydrolates revealed that FH stands out as the most promising bioactive source for cosmetic formulations. However, future research addressing antimicrobial activity is necessary to confirm its potential incorporation into dermatological and cosmetic formulations., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

The role of the fungal community, the mycobiota, in the health of the vagina is currently an important area of research. The emergence of new sequencing technologies and advances in bioinformatics made possible the discovery of novel fungi inhabiting this niche. Candida spp. constitutes the most important group of opportunistic pathogenic fungi, being the most prevalent fungal species in vulvovaginal infections. However, fungi such as Rhodotorula spp., Naganishia spp. and Malassezia spp. have emerged as potential pathogens in this niche, and therefore it is clinically relevant to understand their ecological interaction with Candida spp. The main aim of this study was to evaluate the impact of yeasts on Candida albicans ' pathogenicity, focusing on in-vitro growth, and biofilm formation at different times of co-culture and germ tube formation. The assays were performed with isolated species or with co-cultures of C. albicans (ATCC10231) with one other yeast species: Rhodotorula mucilaginosa (DSM13621), Malassezia furfur (DSM6170) or Naganishia albida (DSM70215). The results showed that M. furfur creates a symbiotic relationship with C. albicans , enhancing the growth rate of the co-culture (149.69%), and of germ tube formation of C. albicans (119.8%) and inducing a higher amount of biofilm biomass of the co-culture, both when mixed (154.1%) and preformed (166.8%). As for the yeasts R. mucilaginosa and N. albida , the relationship is antagonistic (with a significant decrease in all assays), thus possibly repressing the mixture's pathogenicity. These results shed light on the complex interactions between yeasts in the vaginal mycobiome.

Candida albicans is the leading cause of vulvovaginal yeast infections; however, other species are becoming relevant in this niche. The spatial distribution of these fungi in the female genital tract remains poorly understood. In this study, swab samples were collected from 33 patients, first from the anterior vulva and then from the upper third and right lateral wall of the vagina: 16 were with symptoms of vulvovaginal candidiasis and 17 were without characteristic symptoms; furthermore, the genus and species of each isolate were identified. In vitro susceptibility testing for fluconazole and clotrimazole was performed for all isolates. Candida albicans was the most common species (63.6%), followed by Rhodotorula spp. (51.5%), and then Candida parapsilosis (15.2%). Rhodotorula spp. and C. parapsilosis were more commonly associated with colonization, and C. albicans with infection. Rhodotorula spp. isolates presented a low susceptibility to fluconazole, with the MIC ranging from 32 to >64 µg/mL. Differences in susceptibility to fluconazole and clotrimazole between the pairs of vaginal and vulvar isolates were found for Candida albicans , Rhodotorula spp., and Nakaseomyces glabratus . The results suggest that different niches may impact the susceptibility profiles of the isolates, as well as their different clinical behaviors.

We aimed to incorporate Thymbra capitata essential oil (TCEO), a potent antimicrobial natural product against bacterial vaginosis (BV)-related bacteria, in a suitable drug delivery system. We used vaginal sheets as dosage form to promote immediate relief of the typical abundant vaginal discharge with unpleasant odour. Excipients were selected to promote the healthy vaginal environment reestablishment and bioadhesion of formulations, while the TCEO acts directly on BV pathogens. We characterized vaginal sheets with TCEO in regard to technological characterization, predictable in vivo performance, in vitro efficacy and safety. Vaginal sheet D.O (acid lactic buffer, gelatine, glycerine, chitosan coated with TCEO 1% w/w ) presented a higher buffer capacity and ability to absorb vaginal fluid simulant (VFS) among all vaginal sheets with EO, showing one of the most promising bioadhesive profiles, an excellent flexibility and structure that allow it to be easily rolled for application. Vaginal sheet D.O with 0.32 µL/mL TCEO was able to significantly reduce the bacterial load of all in vitro tested Gardnerella species. Although vaginal sheet D.O presented toxicity at some concentrations, this product was developed for a short time period of treatment, so this toxicity can probably be limited or even reversed when the treatment ends.

Ethnopharmacological Relevance: Thymus mastichina (L.) L. (TM) and Cistus ladanifer L. (CL) are two Portuguese autochthonous species with traditional skin application in folk medicine. TM is majorly known for its antiseptic and wound healing properties, as an external anti-inflammatory agent and for its application in folk cosmetics and hygiene products. Its use in acne vulgaris has also been reported. CL is traditionally used in remedies for wounds, ulcers and other skin ailments such as psoriasis and eczema. Its application has been found useful due to its anti-inflammatory, astringent, wound healing and antiseptic properties., Aim of the Study: With this work, we aimed to investigate relevant bioactivities related with the traditional application of TM and CL essential oils (EOs) and hydrolates (by-products of EO production) in skin ailments. Specifically their in vitro antioxidant, anti-inflammatory, cytotoxic, wound healing and antimicrobial properties were evaluated. The chemical composition of both EOs and respective hydrolates was also characterized., Materials and Methods: Chemical characterization of EOs and hydrolates was performed by GC-FID and GC-MS. Cellular biocompatibility was evaluated using the MTT assay in macrophages (RAW 264.7) and fibroblasts (L929) cell lines. Anti-inflammatory activity was investigated by studying nitric oxide (NO) production by macrophages with Griess reagent. Wound healing potential was evaluated with the scratch-wound assay. The antioxidant potential was studied by the DPPH scavenging method. Antimicrobial activity was evaluated by broth microdilution assay against relevant microbial strains and skin pathogens, namely Staphylococcus aureus, Staphylococcus epidermidis, Cutibacterium acnes, Pseudomonas aeruginosa, Escherichia coli, Candida albicans and Aspergillus brasiliensis., Results: The major compounds present in TM and CL EOs were 1,8-cineole and α-pinene, respectively. 1,8-cineole and E-pinocarveol were the major compounds in the correspondent hydrolates. CL EO presented the highest anti-inflammatory potential [EC 50  = 0.002% (v/v)], still with significant cytotoxicity [IC 50  = 0.012% (v/v)]. TM preparations presented anti-inflammatory potential, also presenting higher biocompatibility. The same profile was present on fibroblasts regarding biocompatibility of the tested preparations. CL EO and hydrolate increased fibroblasts' migration by 155.7% and 148.4%, respectively. TM hydrolate presented a milder activity than CL hydrolate, but wound healing potential was still present, increasing cell migration by 125.1%. All preparations presented poor antioxidant capacity. CL EO presented higher antimicrobial activity, with MICs ranging from 0.06% (v/v) to 2% (v/v), against different microorganisms., Conclusions: Anti-inflammatory and skin repairing potential were present for CL preparations. TM hydrolate presented an interesting biocompatible profile on both cell lines, also presenting anti-inflammatory potential. Furthermore, EOs from both species presented antimicrobial activity against a panel of different microorganisms. These in vitro bioactivities support some of their traditional skin applications, specifically regarding their antiseptic, wound healing and anti-inflammatory uses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Despite the vaginal mucosa is able to respond to allergenic stimuli, vaginal allergic responses have been under investigated in clinical practice. Thus, we aimed to identify the most frequent etiological agents responsible for vulvovaginal allergies, the prevalent signs/symptoms, and the diagnostic tests applied in this clinical condition., Methods: Literature search was performed on PubMed, Scopus, Scielo, Web of Science, and EMBASE. The study protocol was registered on PROSPERO (CRD42020167238). Studies were divided in two groups depending on allergen exposure route. Due to a significant number of studies correlating allergy to Candida infection, subgroup analysis was included., Results: In direct exposure cases, Human Seminal Plasma was the most prevalent allergen, sensitizing 73% of affected women. These women presented localized swelling and burning as prevalent symptoms, affecting 42/68 and 36/68 women, respectively. Cutaneous Prick tests were applied in 58/68 women, either alone or combined with IgE measurements. Regarding cases of indirect/unidentified exposure, house dust mites was the most prevalent allergen (54%), followed by pollen (44%). Predominant symptoms were vulvar pruritus and burning, affecting 67/98 and 52/98 women. Skin prick test was the most prevalent diagnostic method used among different studies. Hypersensitivity toward Candida antigen was present in only half (163/323) of women presenting concomitant allergy and Candida infection., Conclusion: From the two types of allergen exposure that can cause vulvovaginal allergic responses, direct contact of the antigen with the vulva and/or vagina was the most prevalent. Still, allergens can also sensitize the vaginal mucosa secondarily to other exposure route, specifically aeroallergens., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: The human papillomavirus (HPV) is responsible for over 90% of all cervical cancer cases. The use of vaginal gels is often indicated for local vaginal drug delivery. Previous studies have shown that Thymus vulgaris essential oil (TEO) exhibits anticancer properties besides antifungal and antibacterial properties. Its activity derives from a specific increase in free radicals and oxidative stress caused in cancer cells. Furthermore, mitoxantrone (MTX), an anthracenedione, and C 8 , an acridine orange derivative, were shown to inhibit the growth of the cervical cancer cell line HeLa., Results: The results showed that TEO + C 8 is the most promising formulation in terms of viscosity and osmolality properties in vaginal fluid simulant (VFS). The combined action of TEO with the compounds MTX and C 8 resulted in HeLa cell viability reduction compared with the effect obtained with the individual formulations containing each one of the compounds., Conclusions: The formulation TEO + C 8 holds promise in terms of cost-benefit and topical application of the active compound for the HeLa cells., Competing Interests: Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

The use of in vitro human skin permeation tests is of value when addressing the quality and equivalence of topical drug products in Europe and the US. Human skin is the membrane of choice for these studies. The use of human skin as a membrane is hindered by limited access, high variability of results, and limited applicability for drugs with low skin permeability. Reconstructed human epidermis (RhE) models are validated as skin surrogates for safety tests and have been explored for percutaneous absorption testing. Clotrimazole poorly permeates human skin and is widely available for topical treatments. In this study, clotrimazole creams were used to test the ability of RhE to be used as biological membrane for bioequivalence testing, based on the Draft Guideline on Quality and Equivalence of Topical Products (CHMP/QWP/708282/2018) using a discriminative and modified in vitro permeation test (IVPT). To fulfill the validation of a discriminatory method, Canesten ® 10 mg/g cream was compared with a test product with the same drug strength, along with two "negative controls" dosed at a 50% and 200% drug strength. Products were compared in finite dose conditions, regarding maximal flux (J max ) and the total amount of drug permeated ( A total ). The results showed the discriminatory power of the method among the three drug strengths with no interference of the placebo formulation. The study design and validation complied with the requirements established in the guideline for a valid IVPT. This new test system allowed for the equivalence comparison between test and comparator product. Higher permeability of the RhE compared to human skin could be observed. This arose as a strength of the model for this modified IVPT bioequivalence testing, since comparing permeation profiles among products is envisaged instead of drawing absolute conclusions on skin permeation extent. These results may support the acceptance of RhE as biological membranes for modified IVPT in bioequivalence testing of topical products.

Dermatophagoides pteronyssinus and Dermatophagoides farinae are the most common House Dust Mite (HDM) species in home environments worldwide and responsible for HDM allergy. Since the prevalence of HDM-related clinical conditions is linked to exposure to the mite itself, the detection of HDM in the human households gains importance. We aimed to develop a fast and accessible multiplex PCR to detect and distinguish two relevant HDM species in house dust. New primers were designed, and sensitivity analysis was performed. Sequencing of PCR products was also performed to confirm the method's specificity. The limit of detection of the multiplex PCR for both species was as low as 30 pg µL -1 . The application of the multiplex PCR to dust samples also resulted in the identification of both species with high sensitivity. The protocol required small amount of template, reagents and a short reaction time thus presenting an alternative to classically used techniques for HDM identification.

Previous studies have revealed that Candida albicans isolates involved in chronic vulvovaginal candidosis (cVVC) phenotypically express less virulent traits than clinical isolates involved in sporadic infections. In this study, we aimed to further explore this finding by studying the behaviour of those same clinical isolates in in-vitro models of infection. Eighteen clinical Candida albicans isolates were collected from women suffering sporadic (eight isolates) or chronic infections (ten isolates). Adhesion to HeLa cells (human cervical cancer epithelial cell line) and resistance to phagocytosis by RAW 264.7 cells (murine macrophages cell line) were tested in-vitro. In addition, phenotypic expression of virulence factors related with either adhesion or resistance to phagocytosis was tested in-vitro. Results indicated that yeast isolates involved in sporadic infection adhered in a higher proportion of HeLa cells than those of chronic infections, which was related with their ability to produce biofilm (p < 0.05). The ability to evade phagocytosis was related to an elevated production of proteases (p < 0.05) by chronic isolates, while sporadic isolates’ resistance to phagocytosis was related to a higher hydrophobicity of cell walls (p < 0.05). We conclude that the evasion of macrophage-mediated phagocytosis related to the production of proteases might be an important factor involved in the recurrence of vulvovaginal candidosis infection.

Ethnopharmacological Relevance: Thymus × citriodorus (Pers.) Schreb. is an interspecific hybrid between Thymus pulegioides and Thymus vulgaris, known for its pharmacological activities as diaphoretic, deodorant, antiseptic and disinfectant, the last mostly related with its antimicrobial activity. The folk use of other extracts, as hydrolates, have also been disseminated, as regulators of oily skin with anti-acne effect., Aim of the Study: We aimed to evaluate the anti-acne potential of two Thymus x citriodorus (TC) preparations, the essential oil (EO) and the hydrolate, to be used as active ingredients for skin applications. Specifically, we intend to validate their anti-acne potential by describing their activity on acne related bacteria, bacterial virulence, anti-oxidant and anti-inflammatory potential, and biocompatibility on inflammatory cells. Additionally, we aimed to report their ecotoxicity under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), thus focusing not only on the consumer, but also on environmental safety assessment., Materials and Methods: Minimum inhibitory concentration (MIC) against C. acnes, S. aureus and S. epidermidis was evaluated. Minimum lethal concentration (MLC) was also determined. The effect on C. acnes biofilm formation and disruption was evaluated with crystal violet staining. Anti-inflammatory activity was investigated on LPS-stimulated mouse macrophages (RAW 264.7), by studying nitric oxide (NO) production (Griess reagent) and cellular biocompatibility through MTT assay. In-vitro NO and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging potential were also evaluated. The ecotoxicity was evaluated using Daphnia magna acute toxicity assays., Results: EO presented direct antimicrobial activity, with visual MICs ranging from 0.06% for S. epidermidis and C. acnes to 0.125% for S. aureus. MLCs were higher than the obtained MICs. Hydrolate revealed visual MIC only for C. acnes. TC essential oil was effective in preventing biofilm formation and disrupting preformed biofilms even at sub-inhibitory concentrations. Hydrolate showed a more modest anti-biofilm effect. Regarding anti-inflammatory activity, TC hydrolate has a higher cellular biocompatibility. Still, both plant preparations were able to inhibit at least 50% of NO production at non-cytotoxic concentrations. Both EO and hydrolate have poor anti-oxidant activities. Regarding the ecotoxicity, TC essential oil was classified under acute 3 category, while the hydrolate has proved to be nontoxic, in accordance to the GHS., Conclusions: These results support the anti-acne value of different TC preparations for different applications. TC hydrolate by presenting higher biocompatibility, anti-inflammatory potential and the ability to modulate C. acnes virulence, can be advantageous in a product for everyday application. On the other hand, EO by presenting a marked antimicrobial, anti-biofilm and anti-inflammatory activities, still with some cytotoxicity, may be better suited for application in acute flare-ups, for short treatment periods., (Copyright © 2021 Elsevier B.V. All rights reserved.)

To use or not to use, that is the first decision to take regarding a drug product. This mandatory step for adherence dictates product efficacy. The determinants for such decision do not only rely on the priority of the therapeutic or preventive strategy, but are related to a complex network of perceptions, preferences, personal and cultural backgrounds, and results from previous experiences. Women's preferences for dosage forms and even for drug delivery routes have been mainly studied in the fields of contraception and HIV prevention (and their related multipurpose approaches). Much less attention has been devoted to other therapeutic or preventive strategies. In a time when patient-centred approaches and shared decisions are increasingly valued, considering women's preferences and their main determinants is essential for product development and selection. Such products will be more likely to be chosen and used as intended, increasing efficacy, and reducing the overall costs related with these treatments. This knowledge shall be integrated in early stages of product development. This article reviews the state of the art related with women's preferences and acceptance for different dosage forms and drug delivery routes involved in women's health. The methodologies used for collecting these data and their major drawbacks are discussed. Results obtained from acceptability studies and the main determinants for selection of preventive and treatment drug products are discussed as tools for new developments in the field., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Chronic vulvovaginal candidosis results either from reinfection or from the ability of Candida spp. to persist in the vulva and/or vagina. Persistence is usually associated with increased antifungal (mainly azoles) resistance rates, which can explain treatment failure, and/or increased expression of virulence factors by Candida spp. The aim of this study was to assess the mechanisms leading to Candida spp persistence, by studying sequential isolates from women with chronic vulvovaginal candidosis, focusing on strains genotypes, azole resistance, and ability to form biofilms along the period of clinical evaluation. The strains were identified at species level by automated analysis of biochemical profiles and molecular typing evaluated by polymorphic DNA analysis. The capacity to form biofilm was assessed with a microtiter plate assay. Fluconazole susceptibility was determined by the microdilution broth assay at both pH 7 (following the recommended guideline) and pH 4.5 (as representative of vaginal pH). We studied samples from 17 clinically recurrent cases. In 53% of the chronic cases there were two or more isolates that had a phylogenetic relationship while the remaining (47%) were caused by different species. In those cases where related strains were involved in recurrence, we verified an increase in MIC at pH 7 and also an increased capacity to form biofilms over time. Significant correlation between these two parameters was observed only in cases caused by C. glabrata, evidencing the importance of these two factors to enhance persistence in the vaginal mucosa for this particular species., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Introduction: The vast majority of the species of the genus Candida spp. is commensal in humans; however, some are opportunistic pathogens that can cause infection, called candidosis. Among the different types of candidosis, we highlight the vulvovaginal (VVC) which can occur in two main clinical variants: chronic (cVVC) and episodic or sporadic. The incidence of cVVC has been worrying the scientific community, promoting the research on genotypic and phenotypic causes of its occurrence. We summarize important findings on factors that favor chronic vulvovaginal candidosis with respect to molecular epidemiology and the expression of various virulence factors, while clarifying the terminology involving these infections., Aim and Methodology: The aim of this review was to gather research that linked virulence factors to VVC and its persistence and recurrence, using two databases (Pubmed and Google Scholar). Predisposing factors in women for the occurrence of cVVC and some studies that refer new preventive and alternative therapies were also included, where appropriate., Results and Discussion: Several studies have been shedding light on the increasing number of persistence and recurrences of VVC. The expression of virulence factors has been related to both chronic forms of VVC and antifungal resistance. Other studies report mutations occurring in the genome of Candida spp. during the infection phase which may be important indications for new therapies. The introduction of preventive therapies and new therapies has revealed great importance and is also highlighted here., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Cervical cancer is one of the most common cancers and is one of the major cause of deaths in women, especially in underdeveloped countries. The patients are usually treated with surgery, radiotherapy, and chemotherapy. However, these treatments can cause several side effects and may lead to infertility. Another concerning gynecologic cancer is endometrial cancer, in which a high number of patients present a poor prognosis with low survival rates. AS1411, a DNA aptamer, increases anticancer therapeutic selectivity, and through its conjugation with gold nanoparticles (AS1411-AuNPs) it is possible to improve the anticancer effects. Therefore, AS1411-AuNPs are potential drug carriers for selectively delivering therapeutic drugs to cervical cancer. In this work, we used AS1411-AuNPs as a carrier for an acridine orange derivative (C 8 ) or Imiquimod (IQ). The AS1411 aptamer was covalently bound to AuNPs, and each drug was associated via supramolecular assembly. The final nanoparticles presented suitable properties for pharmaceutical applications, such as small size, negative charge, and favorable drug release properties. Cellular uptake was characterized by confocal microscopy and flow cytometry, and effects on cellular viability were determined by MTT assay. The nanoparticles were then incorporated into a gel formulation of polyethylene glycol, suitable for topical application in the female genital tract. This gel showed promising tissue retention properties in Franz cells studies in the porcine vaginal epithelia. These findings suggest that the tested nanoparticles are promising drug carriers for cervical cancer therapy.

Nanoparticles offer targeted delivery of drugs with minimal toxicity to surrounding healthy tissue and have great potential in the management of human papillomavirus (HPV)-related diseases. We synthesized lipid-modified AS1411 aptamers capable of forming nanoaggregates in solution containing Mg 2+ . The nanoaggregates presented suitable properties for pharmaceutical applications such as small size (100 nm), negative charge, and drug release. The nanoaggregates were loaded with acridine orange derivative C 8 for its specific delivery into cervical cancer cell lines and HPV-positive tissue biopsies. This improved inhibition of HeLa proliferation and cell uptake without significantly affecting healthy cells. Finally, the nanoaggregates were incorporated in a gel formulation with promising tissue retention properties aiming at developing a local delivery strategy of the nanoaggregates in the female genital tract. Collectively, these findings suggest that the nanoformulation protocol has great potential for the delivery of both anticancer and antiviral agents, becoming a novel modality for cervical cancer management., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Recurrent vulvovaginal candidiasis (RVVC) is caused by Candida spp., a vaginal colonizer. Despite the clinical importance of RVVC, little is known regarding the characteristics of the disease in Portugal. Thirty-six clinical cases were analyzed, comprising 93 yeast vulvovaginal isolates obtained from women attending a gynecologic consultation at a private clinic. Of these, 18 women were diagnosed with RVVC, while other 18 women had a sporadic episode of infection (nonrecurrent vulvovaginal candidiasis [NR-VVC]). Species identification was performed with CHROMagar chromogenic medium and by analysis of biochemical profiles. In addition, antifungal susceptibility testing for two azole compounds was performed by broth microdilution. We found that Candida albicans was isolated from both NR-VVC and RVVC cases, being highly predominant; C. glabrata and C. tropicalis were also isolated. Resistance to at least one antifungal was detected in up to 65% of the isolates, and resistance to both antifungals reached a frequency of 25%. Moreover, azole-resistant isolates were distributed among all species identified. We conclude that in the studied group of patients, C. albicans is in fact the major player both in NR-VVC and in RVVC, C. glabrata being more frequently associated with recurrence ( p  < 0.05). In addition, we found a high proportion of azole-resistant strains.

Bacterial vaginosis (BV) is the most frequent vaginal infection worldwide. It is caused by the overgrowth of anaerobic vaginal pathogens such as Gardnerella spp. BV has been associated with the occurrence of dense multispecies biofilms on the vaginal mucosa. Treatment of biofilm-associated infections such as BV is challenging. In this study, we have tested the role of a quaternary ammonium compound, dequalinium chloride (DQC), in the eradication of Gardnerella spp. biofilms. The effects of the test substance on the biomass and the metabolic activity of the biofilm of Gardnerella spp. were assessed in vitro using a microtiter plate assay. In addition, the effect of DQC on the Gardnerella spp. biofilm was further assessed by using scanning electron microscopy and confocal laser scanning microscopy. The results showed that DQC was particularly effective in the destruction of BV-associated Gardnerella spp. biotypes, impacting both their biomass and metabolic activity. In addition, the disruption of biofilm architecture was evident and was probably caused by multiple mechanisms of action. We conclude that DQC is an antibiofilm agent and is able to efficiently destroy Gardnerella spp. BV-associated biofilms. Therefore, it is a valid option for BV therapy and has the potential to prevent BV recurrences.

Vulvovaginal candidosis (VVC), caused mainly by the yeast Candida albicans, is the second most prevalent vaginal infection. It has been found to have a large impact on women's quality of life, self-esteem and routines. The prevalence of recurrent vulvovaginal candidosis (RVVC) remains high so the development of alternative treatments is needed. The main objective of this study was to develop and characterize sodium bicarbonate gels to treat VVC. We described key formulation characteristics and analyzed their influence on in vitro performance evaluations. The potential to inhibit Candida albicans's growth, the pH, osmolality, viscosity and rheological performance in contact with vaginal fluid simulant and the bioadhesion's profile (using a vaginal ex vivo porcine model) were studied for all formulations. Among the formulations, formulation C (5% sodium bicarbonate, 1% carbomer and 94% water) was the most effective in inhibiting the C. albicans' growth. This gel presented the same potential (the same MIC 2.5%) to inhibit other etiological agents of VVC (C. glabrata, C. krusei, C. tropicalis and C. parapsilosis) for all species tested. Additionally, sensorial characteristics of gel C were in accord with users' preferences. This gel exhibited physicochemical characteristics acceptable for short term treatments, suggesting good overall performance for the treatment of VVC. Furthermore, Gel C was biocompatible with the HeLa cell line (MTT assay) and was classified as a non-severe irritant in the HET-CAM assay (irritation score 4 ± 1). Overall, gel C was deemed the best performing of the set tested, and suitable for further development., (Copyright © 2020. Published by Elsevier B.V.)

Objective: Vulvovaginal candidosis (VVC) is a condition that impacts the quality of life of women worldwide. At least 5-8% of all VVC cases re-occur. Recurrent vulvovaginal candidosis (RVVC) can be defined as the occurrence of a VVC episode at least four times per year. The reasons for recurrence to occur are poorly understood. This work aims to identify key phenotypic traits associated with RVVC Candida spp. isolates that might be used to plan strategies to control RVVC., Methods: The capacity to form biofilms (with the microtitration plate assay), to develop germinative tube in the presence of fetal bovine serum and to produce phospholipase (in the egg-yolk plate assay) was assessed for a collection of Candida spp. isolates obtained from 17 women diagnosed with RVVC and 16 women with non-recurrent VVC (VVC). The differences obtained regarding the proportion of isolates expressing each virulence factor was assessed by statistical analysis (χ 2 )., Results: We found that C. albicans isolates had a higher ability to form germinative tubes than RVVC isolates (29% vs 4%, p < 0.05). In addition, the ability of Candida spp. isolates to form biofilm (63% vs 51%) and to produce phospholipase (13% vs 11%) was also higher, though not statistically different (p > 0.05)., Conclusions: We conclude that biofilm formation and phenotypic-switching associated with germinative tube production are particularly important C. albicans virulence factors for acute, sporadic VVC cases., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Thermal therapy has gained popularity over the years, and Portugal is one of the richest European countries in mineral therapeutic waters. The interest in the use of these natural mineral waters (NMW) for dermatologic purposes is continuously growing but there is a lack of scientific studies supporting its health benefits. The study aims to investigate the effect of a silica-rich NMW in skin cell homeostasis using two representative cell lines of the epidermis and dermis, keratinocytes and fibroblasts, respectively, in addition to a macrophage cell line. Mouse skin fibroblasts, macrophages and human keratinocytes were exposed to culture medium prepared with NMW. Cell metabolism (MTT or resazurin assays) and cell proliferation (trypan blue exclusion dye assay) were investigated. Migration (scratch-wound assay) and senescence (β-galactosidase activity assay) of fibroblasts were also studied. Exposure to NMW compromised the cell metabolic state of all the cell lines tested. This impairment was more pronounced in skin keratinocytes (60% reduction) relatively to skin fibroblasts (45% reduction) or macrophages (25% reduction). Proliferation of macrophages was reduced threefold upon exposure to thermal water, compared to controls. No differences were observed in migration between fibroblasts exposed to NMW and controls, while a potentiation of senescence of these cells was observed. Our results shed light in the bioactive effects of a silica-rich NMW supporting its therapeutic use. A reduction in both cell metabolism and proliferation of keratinocytes and macrophages supports the empirical clinical benefits of this NMW in hyperkeratotic conditions, such as psoriasis and atopic dermatitis.

Bacterial vaginosis (BV) affects many women and has a high influence on their self-esteem, being associated with huge discomfort and changes in the routines, especially the sexual life. International guidelines recommend the administration of metronidazole, clindamycin or tinidazole orally or intravaginally as the standard treatment. However, the treatment with these antibiotics is associated with high levels of failure and recurrence rates. These may be associated with antibiotic resistance, the inability to eradicate the polymicrobial biofilms, and failure to reestablish acidic pH and the lactobacillus-dominated commensal flora. Therefore, it is emergent to study alternative strategies to replace or to be combined with standard therapies in order to prevent and treat BV more efficiently. Alternative strategies may include antimicrobial substances (other antimicrobials, antiseptics and natural compounds) or substances that aim to reestablish the physiologic vaginal environment (probiotics, prebiotics and acidifying agents) while improving the local immunity response. Besides, the development of formulation strategies and new dosage forms and drug delivery systems can improve treatment efficacy and overcome some limitations associated with conventional products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aim: Vulvovaginal candidosis (VVC) is the second most common vaginal infection (20-25%), and about 90% of all VVC cases are caused by Candida albicans. Unprotected sexual intercourse has been implicated as one of the risk factors that lead to an outbreak of VVC. To further investigate the relevance of this particular risk factor, in this study, we aim to evaluate the effect of human semen in the promotion of the growth of C. albicans., Methods: The disposable amount of 41 samples of semen obtained from infertility patients were included in this study, with informed consent. The spermogram and physical characteristics of the samples were performed at the Unit; this information was provided with the anonymity of the samples. Samples were inoculated with a calibrated suspension of C. albicans ATCC 10231 in culture media. After the incubation time, C. albicans CFU/mL was determined., Results: We found that semen allowed the growth of C. albicans (4.30 ± 1.00 CFU/mL), but not as much as the culture medium (9.45 ± 1.90 CFU/mL). Interestingly, we found that the increase in viscosity impaired significantly C. albicans growth. In addition, in what respects to the rate of multiplication of C. albicans in semen, we observed two different trends. However, we found no relation between these and the physical characteristics of the semen samples in which these behaviors were differently observed., Conclusion: Semen has the ability to sustain C. albicans growth, but further studies are needed to elucidate its role in VVC., (© 2020 Japan Society of Obstetrics and Gynecology.)

The common therapeutic indications of Portuguese Natural Mineral Waters (NMWs) are primarily for respiratory, rheumatic and musculoskeletal systems. However, these NMWs have been increasingly sought for dermatologic purposes. Opposing to what is observed in the major European Thermal Centres, there are few scientific evidences supporting the use of Portuguese NMWs for clinical applications. The aim of this study was to characterize the antimicrobial profile of individual NMWs from the central region of Portugal and correlate the results with their physicochemical characterization. An extensive multivariate analysis (principal component analysis) was also performed to further investigate this possible correlation. Six collection strains representing skin microbiota, namely Staphylococcus aureus, Escherichia coli, Corynebacterium amycolatum, Candida albicans, Staphylococcus epidermidis and Cutibacterium acnes, were analysed, and their antimicrobial profile was determined using Clinical and Laboratory Standards Institute M07-A10, M45-A2, M11-A6 and M27-A3 microdilution methods. Different NMWs presented different antimicrobial profiles against the strains used; the physicochemical composition of NMWs seemed to be correlated with the different susceptibility profiles. Cutibacterium acnes showed a particularly high susceptibility to all NMWs belonging sulphurous/bicarbonated/sodic ionic profile, exhibiting microbial reductions up to 65%. However, due to the complex physicochemical composition of each water an overall conclusion regarding the effect of a specific ion on the growth of different microorganisms is yet to be known.

Objective: To develop and characterize a new form of vaginal film. Significance: This formulation is intended to overcome some known limitations of traditional dosage forms. It has an absorptive intention to control symptoms and to improve the treatment of vaginal infections characterized by excessive fluid. The vaginal sheet is a thick drug delivery system easy to manipulate, nontoxic and composed by biocompatible macromolecules and polymers, such as gelatin and chitosan. Methods: The sheets were prepared by formulating gelatin or chitosan based gels isolated or in combination, in association with a plasticizer. Gels were subsequently lyophilized. Different proportions of polymer:plasticizer were tested. Lactose was used as a surrogate to study powder incorporation in the formulation. All formulations were analyzed regarding their organoleptic characteristics, texture (hardness and resilience), in vitro absorption efficiency of vaginal fluid simulant - VFS (pH 4 and 5), pH and acid-buffering capacity. Results: Different properties were obtained by varying polymer and plasticizer proportions. Combinations including gelatin with propylene glycol showed the best organoleptic characteristics. The best proportions were 4:3 and 4:5. Up to 10% of powder was successfully incorporated in the formulation. Hardness and resilience of formulations were largely dependent on the concentration of plasticizer. Absorption of vaginal fluid was found to be highly efficient, especially at pH 5. Buffering capacity, upon dilution in normal saline and VFS, was generally higher for VFS pH 4. Conclusions: The vaginal sheet is a promising solid drug delivery system able to further incorporate drugs to treat vaginal clinical conditions characterized by excessive fluid.

Preclinical safety assessment of vaginal products includes cytotoxicity assays upon cell lines. Furthermore, tissue explants have been considered for application on ex vivo models. In this study, traditional and renewed methods were studied for toxicity assessment of vaginal semisolids upon products currently used in clinical practice as antimicrobials (Gino-Canesten ® , Sertopic ® , Dermofix ® , Gyno-Pevaryl ® , Lomexin ® , Gino Travogen ® , Dalacin V ® ), containing estrogens (Ovestin ® , Blissel ® , Colpotrophine ® ), and reference formulations (Replens ® , Universal Placebo). Two in vitro cytotoxicity tests were performed: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake upon uterine (HEC-1A), cervical (HeLa) and vaginal (VK2 E6/E7) cells, according to ISO/EN 10993-5 (in vitro evaluation of medical devices). Similarly, a strategy to determine tissue viability on ex vivo porcine vaginal model (through MTT reduction assay and histological analysis) was developed and optimized. The vaginal cell line VK2 E6/E7 conducted to the most accurate calculation of half-maximal toxic concentration among all cells on the MTT assay. However, it was shown not be sensitive to the neutral red uptake assay. Tissues from the porcine model were collected with approx. 15% variability in thickness and variation coefficients lower than 25% when testing negative and positive controls were achieved. These models can improve cost-efficiency in early steps of product development., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Lactobacillus sp. are well-known colonizers of human mucosa and frequently used as probiotics. Accurate species identification is crucial both for fundamental studies and biotechnology applications; however, it has been thus far challenging. The aim of this work was to develop a one-step multiplex-PCR assay for detection of ten Lactobacillus species (L. jensenii, L. fermentum, L. acidophilus, L. crispatus, L. reuteri, L. iners, L. casei, L. gasseri, L. plantarum, L. rhamnosus) directly in complex bacterial genomic DNA. A multiplex-PCR assay was optimized based on Box-Behnken experimental design, which showed to be efficient for optimization of all crucial reaction components. Nineteen Lactobacillus strains, including six collection strains and thirteen human isolates were used in order to verify the specificity and sensitivity of the assay. In addition, a set of PCR adjuvants was introduced to remove non-specific amplifications and enhance reaction yield. Among them, Triton™ X-100, Tween ® 20, BSA, and dithiothreitol showed beneficial effects when compared with other adjuvants. The application of the developed method to samples that resulted from the mixing of DNA from the ten strains, resulted in amplicons of the expected sizes (from about 100 to 1000 bp). The detection limit was 1.25 ng/μl for all species with the exception of L. gasseri (0.31 ng/μl). In order to confirm the method applicability on human samples, ten vaginal fluids were enrolled in this study showing that the method can be successfully used on these biological materials. The proposed multiplex-PCR assay was shown to be selective, sensitive and efficient for detection of ten Lactobacillus species directly in human vaginal samples. This method provides a cost-effective and accessible methodology applicable to the detection of Lactobacillus species to different environments. At the same time, this approach represents a considerable improvement over other PCR-based approaches for identification of these species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As an opportunistic fungal pathogen Candida spp. has the ability to form biofilms. The most prescribed drugs for Candida infections, azoles, have shown to be less effective when biofilms are present. In addition, increasing treatment costs and the fact that most prescribed antifungal drugs have only fungistatic activity justify the search for new treatment strategies. One promising approach is third generation antidepressants, selective serotonin re-uptake inhibitors (SSRIs), because of their proven antifungal activity against several Candida spp. Thus, the aim of this work was to determine the ability of two commonly used SSRIs, fluoxetine and sertraline, to impair both biofilm metabolic viability and biofilm biomass. The in vitro effect of fluoxetine and sertraline was individually tested against biofilm metabolic viability and biofilm biomass using the MTT assay and the Crystal Violet assay, respectively. For both drugs, a dose-dependent reduction on both biofilm metabolism and biomass was present. At high concentrations, fluoxetine was able to reduce biofilm metabolism by 96% (C. krusei) and biofilm biomass by 82% (C. glabrata), when compared to the control. At similar conditions, sertraline achieved a reduction of 88% on biofilm biomass (C. glabrata) and 90% on biofilm metabolism (C. parapsilosis). Moreover, fluoxetine showed interesting anti-biofilm activity at previously reported planktonic MIC values and even at sub-MIC values. These results reinforce the potential interest of SSRIs as anti-biofilm agents to be study to counteract resistance phenomena on candidosis.

The HET-CAM (Hen’s Egg Test-Chorioallantoic Membrane) assay is an in vitro alternative to the in vivo Draize rabbit eye test. This qualitative method assesses the irritancy potential of chemicals. The chorioallantoic membrane responds to injury with an inflammatory process similar to that in the rabbit eye’s conjunctival tissue. Regarding topical toxicity assessment of medical devices, ISO 10993-10 states that any skin or eye irritant material shall be directly labelled as a potential vaginal irritant without animal testing, suggesting that the irri­tation potentials for the eye and the vaginal epithelia are similar. The aim of this work was to apply the HET-CAM assay to test the irritancy potential of vaginal formulations. Vaginal semisolid medicines and lubricants currently marketed were tested along with the Universal Placebo formulation that has been shown to be clinically safe. Nonoxynol-9 (N-9), a known vaginal irritant, was enrolled as positive control (concentrations ranging from 0.001 to 100% (v/v)). The assay was conducted according to the ICCVAM – Recommended Test Method (NIH Publication No. 10-7553 – 2010). Formulations were then classified according to irritation score (IS), using the analysis methods (A) and (B). The studied vaginal formulations showed low potential for irritation. N-9 was classified as a severe irritant at concentrations above 2%, which is in line with clinical data, envisaging a possible in vitro/in vivo correlation. IS (B) was considered a more detailed classification output. Although still requiring further validation, the HET-CAM assay seems an ideal prospect for in vitro vaginal irritancy testing.

Vaginal semisolid products are frequently used to treat vaginal infections and atrophy-related symptoms of menopause. Formulations composition and the methods for their characterization, especially those developed concerning the target epithelia, are key tools to predict in vivo results at early stages of product development. However, recent studies on this subject have been almost exclusively focused on anti-HIV preparations. The aim of this work consists on improving traditional characterization methods by using physiological parameters in order to construct predictive tools to characterize a new ideal vaginal semisolid formulation whatever target it may have. Ten vaginal antimicrobial and hormonal products already available in the market were studied (Gino-Canesten®, Sertopic®, Dermofix®, Gyno-pevaryl®, Lomexin®, Gino Travogen®, Dalacin V®, Ovestin®, Blissel®, Colpotrophine®). Furthermore, Universal Placebo gel and Replens® were used for comparison. Products were characterized in terms of: pH and buffering capacity in a vaginal fluid simulant (VFS); osmolality - directly and upon dilution in VFS; textural parameters (firmness, adhesiveness and bioadhesion) using vaginal ex vivo porcine epithelium; and viscosity (including VFS dilution at 37°C and after administration on an ex vivo model). Interestingly, the majority of the tested commercial vaginal formulations did not present technological characteristics close to the ideal ones when tested under target biological conditions. The inclusion of such methodologic adaptations is expected to optimize cost-efficiency of new formulations development by predicting efficacy and safety profiles at early stages of product development., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A study of compounding practices among Portuguese community pharmacies from 2008 to 2011 and pharmacists' perspectives concerning compounding was conducted. The retrospective study was based on an online questionnaire developed to gather information on pharmacies characteristics frequency, and type of compounded preparations. Additionally, difficulties, motivations, and pharmacist's perspectives regarding compounding were assessed. Up to 1,450 Portuguese pharmacies were contacted, and 250 completed questionnaires obtained. Statistical analysis was performed using SPSS Version 21. Frequency and cross-tab analysis was used to describe data. Chi-square test was used to assess statistical significant differences between compounding and non-compounding pharmacies' characteristics. Among all pharmacies, 75.2% reported compounding practices, although the majority prepared less than 50 preparations per year, corresponding to less than 10 different formulations. Those pharmacies' with a higher lifetime activity, number of customers, and team members were associated to compounding practices. Dermatological preparations were the most frequently prepared formulations, followed by oral solutions, and otorhinolaryngological preparations. Dermatologists and pediatricians were the most frequent prescribers of compounded medicines. Regarding future perspectives, 51.4% of pharmacists believed that compounding will decrease. However, 79.1% indicated that they will continue to compound, and 70.7% considered that compounded prescriptions should be encouraged. Patient satisfaction (66.1%) and improvement of the pharmacy image (63.8%) were considered the main advantages of compounding services. Compounded medicines are still prepared in the community pharmacy setting to fulfill special patients' therapeutic needs, especially following dermatologists' and pediatricians' prescriptions. Offering compounding services is perceived by pharmacists as an important factor for high-quality pharmacy service and patient satisfaction.

The vagina stands as an important alternative to the oral route for those systemic drugs that are poorly absorbed orally or are rapidly metabolized by the liver. Drug permeation through the vaginal tissue can be estimated by using in vitro, ex vivo and in vivo models. The latter ones, although more realistic, assume ethical and biological limitations due to animal handling. Therefore, in vitro and ex vivo models have been developed to predict drug absorption through the vagina while allowing for simultaneous toxicity and pathogenesis studies. This review focuses on available methodologies to study vaginal drug permeation discussing their advantages and drawbacks. The technical complexity, costs and the ethical issues of an available model, along with its accuracy and reproducibility will determine if it is valid and applicable. Therefore every model shall be evaluated, validated and standardized in order to allow for extrapolations and results presumption, and so improving vaginal drug research and stressing its benefits., (Copyright © 2015 Elsevier B.V. All rights reserved.)