Protective role of Portuguese natural mineral waters on skin aging: in vitro evaluation of anti-senescence and anti-oxidant properties.

Natural mineral waters (NMWs) emerge from the earth as springs and their beneficial therapeutic effect has been empirically recognized in different countries. Portugal has diverse NMW resources that are sought for the relief of different afflictions including dermatological complications. However, there is a lack of scientific validation supporting this empiric knowledge. In this study, we aimed to screen the in vitro bioactivity of Portuguese NMWs with different chemical profiles, namely sulfurous/bicarbonate/sodic (SBS), bicarbonate/magnesium, sulfated/calcic, sulfurous/chlorinated/sodic, sulfurous/bicarbonate/fluoridated/sodic, and chlorinated/sodic, focusing on aging-related skin alterations. Mouse skin fibroblasts and macrophages were exposed to culture medium prepared in different NMWs. Cellular viability was evaluated by MTT assay and etoposide-induced senescence was analyzed through the beta-galactosidase staining kit. Wound healing was investigated by the scratch assay, and phototoxicity/photoprotection after UVA irradiation was evaluated using a neutral red solution. ROS production was quantified using the 2'7'-dichlorofluorescin diacetate dye, and the activity of superoxide dismutase (SOD) was analyzed by a commercial kit after lipopolysaccharide exposure. NMWs within the SBS profile demonstrated anti-senescence activity in skin fibroblasts, along with a variable effect on cellular viability. Among the tested NMWs, two decreased cellular senescence and preserved cell viability and were therefore selected for subsequent studies, together with a SBS NMW with therapeutic indications for dermatologic diseases. Overall, the selected NMW promoted wound healing in skin fibroblasts and activated SOD in macrophages, thus suggesting an anti-oxidant effect. None of the NMWs prevented phototoxicity after UV irradiation. Our results shed a light on the anti-aging potential of Portuguese NMW, supporting their putative application in cosmetic or medical products.

The predictive value of serial serum estradiol and serial endometrial volume on endometrial receptivity on assisted reproductive technology cycles.

BACKGROUND: Diagnosis of endometrial receptivity is still unclear and conflicting. Despite advances in embryo development during assisted reproductive technologies (ART) cycles, the intricate process of implantation is still matter for debate and research. MATERIALS AND METHODS: Prospective case control of 169 subjects during ovarian controlled stimulation for ART. Endometrial receptivity assessment to predict clinical pregnancy with serial continuous biochemical (serum estradiol) and biophysical (endometrial volume and adjusted endometrial volume) parameters were used. Both parameters were compared between negative and positive outcome in terms of clinical pregnancy. RESULTS: No statistical difference was noted between the two groups in terms of demographics and ART procedures and scores. Serum estradiol was significantly higher in the positive group from day 8 after ovarian controlled stimulation. Endometrial volume and adjusted endometrial volume were significantly higher in the positive group as soon as day 6 of ovarian controlled stimulation. CONCLUSIONS: Continuous serum estradiol and 3D endometrial volume and adjusted endometrial volumes may reflect endometrial changes during ART procedures and provide a useful real time tool for clinicians in predicting endometrial receptivity.

Anti-inflammatory potential of Portuguese thermal waters.

In light of Medical Hydrology, thermal waters (TW) are all-natural mineral waters that emerge inside a thermal resort and have therapeutic applications. Their beneficial effect has been empirically recognized for centuries, being indicated for symptom alleviation and/or treatment of several diseases, almost all associated with inflammation. Indeed, an anti-inflammatory effect has been attributed to many different Portuguese TW but there is no scientific validation supporting this empiric knowledge. In the present study, we aimed to investigate the anti-inflammatory properties of 14 TW pertaining to thermal centers located in the Central Region of Portugal, and grouped according to their ionic profile. Mouse macrophage cells stimulated with lipopolysaccharide (LPS), a Toll-like receptor 4 agonist, were exposed to culture medium prepared in TW. Metabolism, nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression levels and the scavenging capacity of TW, were investigated in vitro. 11 out of 14 TW reduced NO production and/or iNOS expression, and/or scavenging activity, in macrophages exposed to LPS. The sulphated/calcic TW did not show any effect on at least one of the inflammatory parameters evaluated. Two sulphurous/bicarbonate/sodic TW and the sulphurous/chlorinated/sodic TW promoted an increase in NO production and/or iNOS expression. Our results validate, for the first time, the anti-inflammatory properties of Portuguese TW, supporting their therapeutic use in the treatment of inflammation-related diseases and promoting their putative application in cosmetic products and medical devices.

Subendometrial resistence and pulsatility index assessment of endometrial receptivity in assisted reproductive technology cycles.

OBJECTIVE: To evaluate Subendometrial and Uterine artery resistance and pulsatility index continuous analysis as a predictor of Endometrial receptivity in Assisted Reproductive Technology (ART) Cycles. DESIGN: Serial 2D transvaginal coloured power doppler ultrasound performed in women on ART cycle to evaluate a pattern that better predicts implantation rates. One hundred sixty-nine subjects on a prospective case control study were assessed. Uterine artery and Subendometrial resistance and pulsatility index was performed to all subjects at baseline (prior to ovarian controlled stimulation), at day 6, 8 and 10 of controlled ovarian stimulation, at trigger day and at embryo transfer day. Also the ratio of fluxometric parameters between Subendometrial blood flow and uterine artery was measured. RESULTS: No statistical difference was noted between two groups in terms of demographics and ART procedures and scores. Uterine artery resistance and pulsatility index showed statistical difference between the two groups (implantation versus non-implantation group). Also statistical significance was obtained between two groups in terms of Subendometrial vascularization. Ratio between Subendometrial and Uterine artery showed lower values of fluxometric parameters in all range for the Subendometrial territory. CONCLUSIONS: Serial Subendometrial and Uterine artery fluxometry may be a useful tool for clinicians in predicting endometrial receptivity enhancing elective embryo transfers in the same ART cycle.

Bacteriocin production of the probiotic Lactobacillus acidophilus KS400.

In the last years, the use of probiotics, including Lactobacillus species, has received much attention to prevent and treat vaginal disorders. These species have been described as having the ability to colonize the epithelial surface and produce antimicrobial metabolites that are able to control the remaining vaginal microflora. This study aimed to identify and characterize, for the first time, a bacteriocin natively produced by Lactobacillus acidophilus KS400 (probiotic strain from Gynoflor®-Medinova AG, Switzerland) and its antimicrobial activity against relevant urogenital pathogens. After organic acids and hydrogen peroxide neutralization in the fermented Lactobacillus acidophilus KS400 culture medium, bacteriocin activity was tested against the indicator microorganism Lactobacillus delbrueckii ATCC9649. The fermentation of Lactobacillus acidophilus KS400 for bacteriocin production was carried out in batch mode, and its antimicrobial activity, optical density and pH were monitored. After production and extraction, the bacteriocin molecular weight was estimated by electrophoresis and tested against vaginal pathogenic microorganisms. As described for other bacteriocins, batch fermentation profiles indicated that bacteriocin production occurs during the exponential growth phase of the lactobacilli, and declines during their stationary growth phase. The molecular weight of the bacteriocin is approximately 7.5 kDa. The bacteriocin containing protein extract was shown to inhibit the growth of Gardnerella vaginalis, Streptococcus agalactiae, Pseudomonas aeruginosa and the indicator strain Lactobacillus delbrueckii ATCC9649. We conclude that L. acidophilus KS400 produces bacteriocin with antimicrobial activity against relevant urogenital pathogens.

Bacterial vaginosis, aerobic vaginitis, vaginal inflammation and major Pap smear abnormalities.

The purpose of this investigation was to evaluate the impact of the vaginal milieu on the presence of abnormal Pap smears and a positive human papilloma virus (HPV) test. A cross-sectional study was conducted between June 2014 and May 2015, evaluating the vaginal discharge by fresh wet mount microscopy and comparing these data with Pap smear findings. Wet mount slides were scored for bacterial vaginosis (BV), aerobic vaginitis (AV), presence of Candida and Trichomonas vaginalis. Cytologic evaluation was done on all Pap smears according to the Bethesda criteria. The cobas© HPV Test (Roche) was performed for HPV detection. A total of 622 cases were evaluated. The mean age of the patients was 41.6 ± 10.65 years (range 21-75). Eighty-three women (13.3 %) had a cytology result worse than low-grade squamous intraepithelial lesion (LSIL). When comparing this group with the one with normal or minor [atypical squamous cells of undetermined significance (ASC-US) or LSIL] Pap smear abnormalities, there were no differences in the presence of Candida (32.5 % vs. 33.2 %, p = 1.0), absence of lactobacilli (38.6 % vs. 32.5 %, p = 0.32) or BV (20.5 % vs. 13.2 %, p = 0.09). On the other hand, moderate or severe inflammation (msI) (41.0 % vs. 28.8 %, p = 0,04), moderate or severe AV (msAV) (16.9 % vs. 7.2 %, p = 0.009) and msAV/BV (37.3 % vs. 20.0 %, p = 0.001) were more common in women with such major cervical abnormalities. No significant association was found between deviations of the vaginal milieu and high-risk HPV infection. The presence of msI or msAV, but not BV, is independently associated with an increased risk of major cervical cytological abnormalities, but not with HPV infection.

Are plant extracts a potential therapeutic approach for genital infections?

More than 40% of the new drugs registered from 1981 to 2006 were obtained, derived or inspired from natural compounds. The influence of natural products in the anti-infective area is quite marked, being a great percentage of drugs derived or extracted from natural products. Vaginal infections are one of the most common reasons a women visits a gynecologist. Given the high popularity of natural therapies among women who suffer from chronic infections, it is urgent for women's healthcare providers to be knowledgeable about such therapies. Additionally, many phytotherapeutic products have been suggested as natural sources of antimicrobial compounds. The increased resistance to conventional antibiotics is one of the main factors justifying the search and development of new antimicrobial agents, especially those of natural origin. Currently, phytochemicals are considered by the scientific community as very attractive targets for potential drug discovery and therapy. In this review, we will focus on the most relevant reports published during the last twenty years about the antimicrobial activity of plant extracts upon microorganisms most frequently involved in genital infections, such as Candida spp., Gardnerella vaginalis, Trichomonas vaginalis and Human papillomavirus. The relationship between their composition and the antimicrobial effects will be highlighted and vaginal therapeutic delivery systems that vehicle plant extracts both commercialized and under investigation will be included.

In vitro assessment of gentian violet anti- candida activity.

BACKGROUND/AIM: Recurrent vulvovaginal candidosis (RVVC) needs alternative therapeutic approaches. Gentian violet (GeV) has been traditionally used to treat mucocutaneous candidosis. The aim of the present study was to evaluate the in vitro activity of GeV against Candida spp. and contribute to clarify the mechanism of action, supporting its clinical therapeutic use. METHODS: Seventeen clinical Candida isolates from RVVC and one C. albicans type collection (ATCC 10231) were studied; the antifungal activity of GeV was evaluated according to the CLSI M27-A3 protocol. To elucidate its mechanism of action, cells were stained with propidium iodide and afterwards analyzed by flow cytometer. RESULTS: GeV showed a fungicidal activity against most Candida spp. C. albicans and C. tropicalis were the most susceptible species. Minimal lethal concentrations were similar to minimal inhibitory concentrations for most tested strains. The fungicidal effect was not related to a primary lesion of the cytoplasmic membrane. CONCLUSION: In accordance with our findings, GeV is a valuable potent fungicidal drug to be used topically, isolated or in combination with oral antifungal drugs, particularly in RVVC cases.

The anti-Candida activity of Thymbra capitata essential oil: effect upon pre-formed biofilm.

ETHNOPHARMACOLOGICAL RELEVANCE: [corrected] Thymbra capitata essential oil is traditionally considered to exhibit powerful antiseptic properties, thus being used to treat cutaneous infections. The aim of the present study was to evaluate the effect of Thymbra capitata essential oil upon pre-formed biofilm of different Candida strains while comparing it with the activity against planktonic cells. MATERIALS AND METHODS: Fifteen Candida isolates were included, corresponding to clinical and collection type strains. Essential oil was obtained by hydrodistillation and its composition analysed by GC/MS. Activity upon planktonic cells was evaluated according to M27-A3 macromethod. Its effect upon 24h preformed biofilm biomass was determined using the crystal violet procedure and the metabolic activity was studied applying the XTT/menadione technique. RESULTS: Biofilm biomass and metabolic activity of all tested species were reduced up to 50% at MIC values. The effect was more pronounced at double MIC values, achieving >80% reduction, except for Candida albicans that presented a more resistant profile (62%). CONCLUSION: Thymbra capitata essential oil presented an important effect upon Candida biofilms. It is proposed as a valuable antifungal product to be used in an appropriate pharmaceutical formulation for the management of resistant mucocutaneous candidosis.

The relationship between Candida species charge density and chitosan activity evaluated by ion-exchange chromatography.

Chitosan, a natural biopolymer presents antifungal activity that seems to be dependent on the interaction of its cationic amino groups and yeast cell surface. In this work we used ion-exchange chromatography to assess the surface charge density of Candida species and subsequently to relate this with their sensitivity profile to chitosan. The ability of several strains from distinct Candida species to interact with strong anionic and cationic exchangers was tested and the yeasts charge surface was assessed by measuring the zeta potential. Our results showed that all the yeast cells tested presented no interaction with the cationic resin and a species-related pattern of interaction was observed with the anionic resin. Specifically, regarding the Q-Sepharose support, Candida glabrata showed the lower retention affinity, followed by Candida albicans, presenting Candida tropicalis an intermediate profile; Candida parapsilosis and Candida guilliermondii revealed a stronger ionic interaction. The yeasts retention synergy in the anionic resin corroborates with the zeta potential outcomes. The behavior observed fit with sensitivity patterns to chitosan as the most susceptible species to chitosan presented higher affinity to the anionic resin in contrast to the less sensitive ones (C. albicans and C. glabrata). This data confirms and reinforces that chitosan activity is probably mediated by an ionic reaction between its amino free groups and ionic charges at the cell surface.

Sodium Tripolyphosphate: an excipient with intrinsic in vitro anti-Candida activity.

Sodium Tripolyphosphate (STPP) is a food additive that is being used in the development of micro and nanoparticles as it induces ionic interactions with chitosan molecules. Although the ability of STPP to inhibit the growth of several food contaminants has been reported, studies on its activity against clinical isolates are scarce. Candida spp. are common causative agents of mucocutaneous infections including the vulvovaginal tegument and new therapeutic approaches are needed in order to treat resistant and recurrent cases. The aim of this study was to evaluate in vitro both antifungal (anti-Candida spp.) activity, and cytotoxicity, on human dermal fibroblasts, of STPP solutions. STPP showed an inhibitory species-dependent activity against several Candida spp. strains being particularly active on C. glabrata, followed by C. guilliermondii. In vitro, STPP showed a concentration dependent cytotoxicity. Therefore STPP use, in low concentrations, seems to be interesting in the development of drug delivery systems for the treatment of vulvovaginal candidosis.

Observational study of vaginal pH in healthy Portuguese women.

AIM: To date, few epidemiological data are available regarding vaginal pH in Portugal. Thus, an epidemiological study was conducted to evaluate vaginal pH in healthy Portuguese women, attending a private gynecologist and its relation with women's socio-demographics, sexual activity and clinical characteristics. METHODS: This was an observational cross-sectional, multicenter and national study in 990 healthy women older than 18 years recruited from September-December 2007. Data regarding socio-demographics, sexual activity (1-5 score ordinal scale: 5 represent the best scenario), physiological status, concomitant treatment and vaginal symptoms were collected. Vaginal pH was measured through specific test strips. RESULTS: The study enrolled women with a median age of 37 years (min-max: 18-83 years) and a median BMI of 24 kg/m2; 63.5% of women practiced some physical activity, 84% were employed, 62.6% were of reproductive age, 22.1% in their post-menopausal phase and 10.4% pregnant. Women considered their sexual lives satisfactory. Median vaginal pH was 4.7 (min-max: 3.5-7.4). Vaginal pH showed positive correlation with age (rs=0.283) and BMI (rs=0.180). Employed women presented a lower median vaginal pH than retired or housewives (4.7 versus 5.6 and 5.3). Higher scores for sexual activity regarding interest, satisfaction, frequency and importance presented lower vaginal pH (respectively rs=-0.171, rs=-0.168, rs=-0.133 and rs=-0.158). Use of contraceptives and pregnancy were associated respectively to lower median levels of vaginal pH. Concomitant treatments and presence of vaginal symptoms were associated with higher median levels of vaginal pH. CONCLUSION: Vaginal pH increases with age, BMI, sexual intercourse without barrier contraception, dryness and irritation symptoms and decreases with physical activity, professional activity and hormone replacement therapy.

Anti-Candida activity of a chitosan hydrogel: mechanism of action and cytotoxicity profile.

Candida spp. are common causative agents of mucocutaneous infections. New therapeutic antifungal drugs are needed to treat chronic disease as these are frequently clinically resistant to azols. Chitosan, among other possible vehicles for active compounds, shows an added value as it appears to have intrinsic antimicrobial properties. The aim of the present study was to evaluate the anti-Candida activity of a medium-molecular-weight chitosan hydrogel (CH), to clarify its possible mechanism of action and to evaluate its cytotoxicity on human fibroblasts. CH antifungal activity was assessed according to CLSI reference M27-A3 protocol; its mechanism of action was investigated by flow cytometry, and its cytotoxicity was studied by MTT assay. CH demonstrated a full inhibition of C. tropicalis, C. krusei, C. guilliermondii and C. parapsilosis growth while impairing C. albicans and C. glabrata viability. Flow cytometry tests showed that CH acts by inducing primary lesion of the cytoplasmic membrane. However, CH showed no cytotoxic effect upon human fibroblasts cells. Resistant strains will require new therapeutic approaches. Chitosan being a good carrier and having itself anti-Candida activity seems to be a promising vehicle to be used for the treatment of mucocutaneous candidosis.

Anti-Candida activity of essential oils.

Anti-Candida activity of essential oils has been widely studied and as a consequence they are being investigated as possible alternatives or complementary therapeutic agents for candidosis. We reviewed the most studied essential oils concerning chemical composition and in vitro/in vivo studies under the perspective of their possible clinical use.

Chemical composition and antifungal activity of the essential oil of Thymbra capitata.

The composition and the antifungal activity of the essential oil of Thymbra capitata on Candida, Aspergillus and dermatophyte strains were studied. Twenty-two samples of the essential oils from the aerial parts of the plant were obtained by hydrodistillation and analysed by GC and GC-MS. All samples are of the carvacrol type, with a high content of carvacrol (60.0 - 65.8 %) and its biogenetic precursors, gamma-terpinene (8.2 - 9.5 %) and p-cymene (6.0 - 7.5 %). The minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) were used to evaluate the antifungal activity against Candida (7 clinical isolates and 3 ATCC type strains), Aspergillus (5 clinical isolates, 2 CECT and 2 ATCC type strains) and 5 dermatophyte clinical strains. To clarify its mechanism of action on Candida strains, the inhibition of germ tube and a flow cytometry assay with propidium iodide (PI) were used. The oil exhibited antifungal activity for all the tested strains, particularly for dermatophytes, with MIC values ranging from 0.08 to 0.32 microL/mL. Regarding Candida, concentrations lower than the MIC values prevented germ tube formation. After a short incubation time the cells incorporated quickly PI, meaning that the fungicidal effect is mainly due to direct lesion of the membrane.

Antifungal activity of Thymus oils and their major compounds.

The increasing recognition and importance of fungal infections, the difficulties encountered in their treatment and the increase in resistance to antifungals have stimulated the search for therapeutic alternatives. Essential oils have been used empirically. The essential oils of Thymus (Thymus vulgaris, T. zygis subspecies zygis and T. mastichina subspecies mastichina) have often been used in folk medicine. The aim of the present study was to evaluate objectively the antifungal activity of Thymus oils according to classical bacteriological methodologies - determination of the minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) - as well as flow cytometric evaluation. The effect of essential oils upon germ tube formation, an important virulence factor, was also studied. The mechanism of action was studied by flow cytometry, after staining with propidium iodide. The chemical composition of the essential oils was investigated by gas chromatography (GC) and gas chromatography/mass spectroscopy (GC/MS). The antifungal activity of the major components (carvacrol, thymol, p-cymene and 1,8-cineole) and also possible interactions between them were also investigated. The essential oils of T. vulgaris and T. zygis showed similar antifungal activity, which was greater than T. mastichina. MIC and MLC values were similar for all the compounds tested. At MIC values of the essential oils, propidium iodide rapidly penetrated the majority of the yeast cells, indicating that the fungicidal effect resulted primarily from an extensive lesion of the cell membrane. Concentrations below the MIC values significantly inhibited germ tube formation. This study describes the potent antifungal activity of the essential oils of Thymus on Candida spp., warranting future therapeutical trials on mucocutaneous candidosis.

Chemical composition and antifungal activity of the essential oil of Origanum virens on Candida species.

The composition and the antifungal activity of the essential oil of Origanum virens on Candida species were studied. The essential oil was obtained from the aerial parts of the plant by hydrodistillation and analyzed by GC and GC-MS. The oil was characterized by its high content of carvacrol (68.1 %) and its biogenetic precursors, gamma-terpinene (9.9 %) and p-cymene (4.5 %). The minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) were used to evaluate the antifungal activity against Candida strains (7 clinical isolates and 3 ATCC type strains). The inhibition of germ tube formation and flow cytometry, using the fluorescent probe propidium iodide (PI), were used to evaluate their mechanisms of action. MIC and MLC values were similar for most tested strains, ranging from 0.16 to 0.32 microL/mL. Concentrations lower than MIC values strongly prevent germ tube formation. The fungicidal effect is primarily due to an extensive lesion of the membrane.

Facts and myths on recurrent vulvovaginal candidosis--a review on epidemiology, clinical manifestations, diagnosis, pathogenesis and therapy.

Approximately three-quarters of all women will experience an episode of vulvovaginal candidosis at least once in their life and 5-10% of them will have more than one attack. Women suffering from three to four attacks within 12 months will be diagnosed with recurrent vulvovaginal candidosis (RVVC). This review covers the large number of proposed aetiological factors for RVVC. The diagnosis of the condition made by conventional means by health providers is often false and is also often misdiagnosed by the affected woman herself. The review covers various methods of diagnosing RVVC and the current knowledge on potential pathogenetic mechanisms proposed for genital candida infections. Treatment of RVVC, including local and systemic antimicrobial therapy and behaviour modification to decrease the risk of recurrences, are discussed. Recent knowledge on drug resistance in candida is also included.

Cytometric approach for a rapid evaluation of susceptibility of Candida strains to antifungals.

OBJECTIVE: To achieve a fast and reliable determination of the susceptibility of Candida strains to amphotericin B (Am B), fluconazole (Flu) and 5-fluorocytosine (5-FC), using cytometric methods as an alternative to the classical dilution method. METHODS: Twenty-three clinical isolates of Candida with different susceptibility patterns were treated for 1 h with two concentrations each of Am B (2 and 8 mg/L), Flu (8 and 64 mg/L) and 5-FC (4 and 32 mg/L), followed by staining with three different fluorochromes, under conditions previously defined through an optimisation study. These were 1 mg/L propidium iodide (PI)/10(6) cells for 30 min at 30 degrees C (a marker that only penetrates cells with severe lesions of the membrane); 0.5 microM FUN-1/10(6) cells for 30 min at 30 degrees C (a fluorescent probe which after entering the yeast cell is converted, by metabolically active yeasts, from a diffuse cytosolic pool with a yellow-green fluorescence into red cylindrical intravacuolar structures) and 0.25 microM of JC-1/10(6) cells for 15 min at 37 degrees C (a monomer that changes reversibly from green to red the J-aggregates, with the increased membrane potential). About 50 000 yeast cells were analysed by flow cytometry (FCM), at FL3 (red, 620 nm) for PI and FL2 (yellow-green, 575 nm) for FUN-1 and the ratio of FL3 to FL1 was determined (red, 620 nm/green, 525 nm) for JC-1; 200 cells of each suspension were also analysed by epifluorescence microscopy (EPM). Viability studies were performed in parallel to count the number of colony forming units. RESULTS: Susceptible (S) strains exposed to Am B and stained with JC-1 showed a dose-dependent decrease in the mitochondrial potential, i.e. a decreased ratio between red/green fluorescence by FCM and a decrease in J-aggregates by EPM. Neither FUN-1 nor PI was useful in the study of Am B activity. Susceptibility to Flu and 5-FC could be detected with FUN-1 staining: metabolic changes were detected by an increase in yellow-green intensity of fluorescence by FCM or a decrease of cylindrical intravacuolar structure formation by EPM, although no decrease in total viability was registered. Staining with JC-1 could predict resistance to both drugs, but did not allow distinction between sensitive dose-dependent strains (S-DD) or intermediate (I) resistance to Flu or 5-FC, respectively, from S strains. PI did not stain Candida cells treated with Flu or 5-FC under our experimental conditions. CONCLUSION: Susceptibility patterns of Candida strains to Am B can be determined by using JC-1, and to Flu and 5-FC by using FUN-1. PI was not a useful probe with which to study the effect of such antifungals under the conditions described here.