A multisite validation of a two hours antibiotic susceptibility flow cytometry assay directly from positive blood cultures.

BACKGROUND: Rapid antimicrobial susceptibility testing (AST) is urgently needed to provide safer treatment to counteract antimicrobial resistance. This is critical in septic patients, because resistance increases empiric therapy uncertainty and the risk of a poor outcome. We validate a novel 2h flow cytometry AST assay directly from positive blood cultures (PBC) by using a room temperature stable FASTgramneg and FASTgrampos kits (FASTinov® Porto, Portugal) in three sites: FASTinov (site-1), Hospital Ramon y Cajal, Madrid, Spain (site-2) and Centro Hospitalar S. João, Porto, Portugal (site-3). A total of 670 PBC were included: 333 spiked (site-1) and 337 clinical PBC (151 site-2 and 186 site-3): 367 gram-negative and 303 gram-positive. Manufacturer instructions were followed for sample preparation, panel inoculation, incubation (1h/37ºC) and flow cytometry analysis using CytoFlex (Site-1 and -2) or DxFlex (site-3) both instruments from Beckman-Coulter, USA. RESULTS: A proprietary software (bioFAST) was used to immediately generate a susceptibility report in less than 2 h. In parallel, samples were processed according to reference AST methods (disk diffusion and/or microdilution) and interpreted with EUCAST and CLSI criteria. Additionally, ten samples were spiked in all sites for inter-laboratory reproducibility. Sensitivity and specificity were >95% for all antimicrobials. Reproducibility was 96.8%/95.0% for FASTgramneg and 95.1%/95.1% for FASTgrampos regarding EUCAST/CLSI criteria, respectively. CONCLUSION: FASTinov® kits consistently provide ultra-rapid AST in 2h with high accuracy and reproducibility on both Gram-negative and Gram-positive bacteria. This technology creates a new paradigm in bacterial infection management and holds the potential to significantly impact septic patient outcomes and antimicrobial stewardship.

Evaluation of rapid colistin susceptibility directly from positive blood cultures using a flow cytometry assay.

Accurate assessment of colistin susceptibility is crucial with the increasing number of multidrug-resistant Gram-negative bacteria and simultaneously increasing colistin resistance. Both EUCAST and CLSI recommend broth microdilution (BMD) to determine colistin susceptibility, however it is cumbersome and growth-dependent. In this study, a rapid flow cytometry method (FASTinovⓇ) to determine colistin susceptibility directly from positive blood cultures (BCs) was evaluated. BCs were spiked with 204 Gram-negative bacilli (137 Enterobacterales, 35 Pseudomonas spp. and 32 Acinetobacter baumannii) at a concentration of 2 × 103 cells/bottle, inoculated with human donor blood and incubated until flagged positive. As quality control strains, two susceptible (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) and two resistant (colistin-resistant mcr-1-positive E. coli NCTC 13846 and Serratia marcescens ATCC 14756) were used. Bacteria were extracted according to assay instructions and were incubated for 1 h at 37 °C with 2 and 4 mg/L colistin and a fluorescent dye, previously optimised. Cells were analysed on CytoFLEX (Beckman Coulter) and AccuriTM C6 Plus (BD Biosciences) flow cytometers. Colistin susceptibility results were automatically provided by BioFAST software (FASTinovⓇ) and compared with those obtained with standard BMD. Overall categorical agreement between this new flow cytometry method and BMD was 99.0%. No very major errors were detected as well as no discrepancies between both flow cytometers. Here we describe a rapid and accurate assay for colistin susceptibility directly from positive BCs with a turnaround time of 2 h versus 48 h required for BMD. This method represents an accurate alternative to standard BMD.

Impact of ERG3 mutations and expression of ergosterol genes controlled by UPC2 and NDT80 in Candida parapsilosis azole resistance.

OBJECTIVES: Candida parapsilosis is a healthcare-related fungal pathogen particularly common among immunocompromised patients. Our understanding of antifungal resistance mechanisms in C. parapsilosis remains very limited. We previously described an azole-resistant strain of C. parapsilosis (BC014RPSC), obtained following exposure in vitro to posaconazole. Resistance was associated with overexpression of ergosterol biosynthetic genes (ERG genes), together with the transcription factors UPC2 (CPAR2-207280) and NDT80 (CPAR2-213640). The aim of this study was to identify the mechanisms underlying posaconazole resistance of the BC014RPSC strain. METHODS: To identify the causative mutation, we sequenced the genomes of the susceptible (BC014S) and resistant (BC014RPSC) isolates, using Illumina technology. Ergosterol content was assessed in both strains by mass spectrometry. UPC2 and NDT80 genes were deleted in BC014RPSC strain. Mutants were characterized regarding their azole susceptibility profile and ERG gene expression. RESULTS: One homozygous missense mutation (R135I) was found in ERG3 (CPAR2-105550) in the azole-resistant isolate. We show that Erg3 activity is completely impaired, resulting in a build up of sterol intermediates and a failure to generate ergosterol. Deleting UPC2 and NDT80 in BC014RPSC reduces the expression of ERG genes and restores susceptibility to azole drugs. CONCLUSIONS: A missense mutation in the ERG3 gene results in azole resistance and up-regulation of ERG genes expression. We propose that this mutation prevents the formation of toxic intermediates when cells are treated with azoles. Resistance can be reversed by deleting Upc2 and Ndt80 transcription factors. UPC2 plays a stronger role in C. parapsilosis azole resistance than does NDT80.

Species distribution and in vitro antifungal susceptibility profiles of yeast isolates from invasive infections during a Portuguese multicenter survey.

This is the first Portuguese multicenter observational and descriptive study that provides insights on the species distribution and susceptibility profiles of yeast isolates from fungemia episodes. Ten district hospitals across Portugal contributed by collecting yeast isolates from blood cultures and answering questionnaires concerning patients' data during a 12-month period. Molecular identification of cryptic species of Candida parapsilosis and C. glabrata complex was performed. The susceptibility profile of each isolate, considering eight of the most often used antifungals, was determined. Both Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocols were applied. The incidence of 240 episodes of fungemia was 0.88/1,000 admissions. Fifteen different species were found, with C. albicans (40 %) being the most prevalent, followed by C. parapsilosis (23 %) and C. glabrata (13 %). Most isolates were recovered from patients admitted to surgical wards or intensive care units, with 57 % being males and 32 % aged between 41 and 60 years. For both the CLSI and EUCAST protocols, the overall susceptibility rates ranged from 74 to 97 % for echinocandins and from 84 to 98 % for azoles. Important resistance rate discrepancies between protocols were observed in C. albicans and C. glabrata for echinocandins and in C. parapsilosis and C. tropicalis for azoles. Death associated with fungemia occurred in 25 % of the cases, with more than half of C. glabrata infections being fatal. The great number of Candida non-albicans is noteworthy despite a relatively low antifungal resistance rate. Studies like this are essential in order to improve empirical treatment guidelines.

Polyethyleneimine and polyethyleneimine-based nanoparticles: novel bacterial and yeast biofilm inhibitors.

Biofilms are commonly involved in medical device-related infections. The purpose of this study was to determine the antimicrobial and anti-biofilm activity of polyethyleneimine (PEI) and PEI-based nanoparticles (nanoPEI) against Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii and Candida albicans (clinical and ATCC strains), and to evaluate their effect upon biofilm formation on polyurethane (PUR)-like catheters. MICs and minimal lethal concentrations of PEI and nanoPEI were determined according to CLSI microdilution reference protocols. For PEI, the MIC value was 195.31 mg l(-1) for all the bacteria and 48.83 mg l(-1) for the yeast strains. For nanoPEI, the MIC value was 1250 mg l(-1) for all the strains except A. baumannii, for which it was 2500 mg l(-1). Biofilm formation was assessed with PUR-like catheter segments and biofilm metabolic activity was quantified by colorimetry with a tetrazolium reduction assay. Plasma membrane integrity and membrane potential were assessed by flow cytometry after staining microbial cells with a membrane-impermeable dye, propidium iodide, and a membrane-potential marker, DiBAC4(3). PEI inhibited growth of all microbial species; higher concentrations of nanoPEI were needed to inhibit growth of all species. Biofilm formation in the presence of anti-bacterial PEI activity was dose-dependent (except for S. epidermidis) and species-related. NanoPEI at 0.5×MIC and MIC significantly reduced the metabolic activity of biofilms of S. aureus, S. epidermidis and A. baumannii, whereas 2×MIC was required in order to inhibit biofilm metabolic activity.

Anti-biofilm activity of low-molecular weight chitosan hydrogel against Candida species.

Candida invasive infections have increased in frequency during the last decades. Such infections are often associated to medical indwelling devices like central venous catheter. The recurrent nature and difficulties in the treatment of these infections are often related to biofilm formation. The objective of this study was to investigate the anti-biofilm activity of low-molecular weight chitosan hydrogel (LMWCH), a natural biopolymer obtained from the N-deacylation of crustacean chitin, upon clinical relevant Candida species. The in vitro ability of LMWCH to impair biofilm formation and to disorganize a preformed biofilm was tested in polystyrene microplates and quantified by the semi quantitative XTT assay and by the crystal violet assay. LMWCH in vivo efficacy as a coating for medical indwelling devices was evaluated for the first time for Candida parapsilosis, using a mouse subcutaneous foreign body model using polyurethane catheter segments. Scanning electron microscopy was used to access biofilm architecture after LMWCH treatment. We found that LMWCH efficiently impaired biofilm formation of all Candida species, also promoting biofilm disaggregation. Most importantly, LMWCH was able to significantly inhibit biofilm formation by C. parapsilosis in an in vivo catheter mouse model. SEM images showed biofilm collapsed cells compatible with membrane damage, suggesting that this could be one of the possible mechanisms underlying biofilm impairment. LMWCH revealed to be a promising compound for treatment of candidiasis or its prevention through medical device coating.

In vivo antibiofilm effect of cerium, chitosan and hamamelitannin against usual agents of catheter-related bloodstream infections.

OBJECTIVES: Catheter-related bloodstream infections (CRBSIs) are common healthcare-associated infections associated with increased morbidity and medical costs. Antiseptic- and antibiotic-coated central venous catheters (CVCs) have been proposed to reduce the incidence of CRBSIs, with variable success. The aim of this study was to determine the in vivo antibiofilm activity of biocompatible and inexpensive compounds, such as cerium nitrate, chitosan and hamamelitannin, against usual agents of CRBSIs. METHODS: The antibiofilm effect of cerium nitrate, chitosan and hamamelitannin was tested against Staphylococcus epidermidis, Staphylococcus aureus, Acinetobacter baumannii and Candida albicans in a mouse foreign body infection model, using polyurethane catheter segments. Biofilm formation was assessed with a crystal violet assay to quantify the total biomass, with a tetrazolium reduction assay to quantify the metabolic activity and with scanning electron microscopy. RESULTS: At subinhibitory concentrations, cerium nitrate significantly reduced biofilm formation by C. albicans, chitosan significantly decreased biofilm formation by S. epidermidis and C. albicans, and hamamelitannin significantly inhibited all bacterial biofilms. DISCUSSION: The in vivo antibiofilm effect of cerium nitrate against C. albicans and of chitosan against C. albicans and S. epidermidis, at subinhibitory concentrations, makes them promising alternatives to coat CVCs. Moreover, the microbicidal effect on a wider range of CVC colonizers was previously reported in vitro for both compounds, at higher concentrations. For all bacterial strains, the highest in vivo antibiofilm efficacy was achieved with hamamelitannin. For A. baumannii, this is the first report of in vivo inhibition.

A novel flow cytometric assay for rapid detection of extended-spectrum beta-lactamases.

The rapid detection of extended-spectrum beta-lactamases (ESBLs) is a challenge for most clinical microbiology laboratories because inaccurate identification of ESBL producers has important clinical implications for both antibiotic treatment and infection control. The aim of our study was to develop a rapid detection assay of ESBL producers based upon flow cytometric analysis. Antimicrobial susceptibility testing followed by molecular characterization of bla(TEM) , bla(SHV) or bla(CTX-M) genes was performed on clinical isolates (41 ESBL positive and 20 ESBL negative) and isolates expressing well-characterized beta-lactamases, including ESBLs (n = 13), plasmid AmpCs (n = 3), oxacillinases (n = 5) and carbapenemases (n = 3). Additionally, two ATCC strains recommended by CLSI for susceptibility testing were used as controls. The flow cytometry analysis protocol involved an incubation of bacterial cells with different concentrations of ceftazidime (1, 2 and 4 mg/L) and cefotaxime (4, 8 and 16 mg/L) for 1 and 2 hours, in the presence and absence of clavulanic acid; subsequently, cells were stained with the fluorescent dye Bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4) (3)], a lipophilic anion able to diffuse across depolarized membranes. Additionally, CFU counts were performed. Susceptible isolates displayed increased fluorescence after 1 hour of incubation; conversely, the increase of the depolarized population was only observed after incubation with clavulanic acid associated with ceftazidime or cefotaxime in ESBL producers. An excellent correlation was obtained between the number of non-depolarized bacteria quantified by flow cytometry and by conventional CFU assays. A novel, accurate and fast flow cytometric assay is available to detect the presence of ESBLs.

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.

Detection of Legionella pneumophila on clinical samples and susceptibility assessment by flow cytometry.

Culture in selective media represents the standard diagnostic method to confirm Legionella pneumophila infection, despite requiring a prolonged incubation period; antigen detection by immunofluorescence (IFS) and molecular techniques are also available, but they do not allow antimicrobial susceptibility evaluation. Our objective was to optimise flow cytometry (FC) protocols for the detection of L. pneumophila in respiratory samples and for susceptibility evaluation to first-line drugs. In order to optimise the FC protocol, a specific monoclonal antibody, conjugated with fluorescein isothiocyanate (FITC), was incubated with type strain L. pneumophila ATCC 33152. The limit of detection was established by analysing serial dilutions of bacterial suspension; specificity was assayed using mixtures of prokaryotic and eukaryotic microorganisms. The optimised FC protocol was used to assess 50 respiratory samples and compared with IFS evaluation. The susceptibility profile to erythromycin, ciprofloxacin and levofloxacin was evaluated by FC using propidium iodide and SYBR Green fluorescent dyes; the results were compared with the Etest afterwards. The optimal specific antibody concentration was 20 µg/ml; 10(2)/ml Legionella organisms were detected by this protocol and no cross-reactions with other microorganisms were detected. The five positive respiratory samples (10 %) determined by IFS were also detected by FC, showing 100 % correlation. After 1 h of incubation at 37 °C with different antimicrobials, SYBR Green staining could discriminate between treated and non-treated cells. A novel flow cytometric approach for the detection of L. pneumophila from clinical samples and susceptibility evaluation is now available, representing an important step forward for the diagnosis of this very relevant agent.

Novel method for evaluating in vitro activity of anidulafungin in combination with amphotericin B or azoles.

A combination of drugs possessing different targets has been used as salvage therapy, although without scientific support. In vitro studies validating such combinations are scarce, and the methodology is very laborious and time-consuming. This study proposes a flow cytometric (FC) protocol as an alternative to evaluate the effect of the combination of anidulafungin (AND) with amphotericin B (AMB) and azoles (fluconazole and voriconazole), tested upon 39 and 36 Candida strains, respectively. The concentration assayed in the combination was 0.5× MIC of each drug. The membrane potential marker DiBAC(4)(3) [Bis-(1,3-dibutylbarbituric acid) trimethine oxonol] was used for AND-AMB, and the metabolic marker FUN-1 was used for AND-azoles. Drug interaction was determined by calculating a staining index (SI): the sum of the percentage of depolarized cells (DC) after treatment with drug combinations divided by the DC of the drug alone, and the sum of the mean intensity of fluorescence (MIF) displayed by cells treated with drug combinations divided by the MIF of the drug alone for FUN-1. An SI of <1 means antagonism, an SI between 1 and 4 means no interaction, and an SI of >4 means synergism. The combination of AND and AMB by FC and checkerboard was synergistic for 46 and 43% of isolates and antagonistic for 5 and 8%, respectively. For the combination of AND and azoles, it was synergistic for 36% and antagonistic for 3% by FC and synergistic for 44% and antagonistic for 3% by checkerboard. When the FC method was compared to the gold standard checkerboard method, the agreement was 0.91 (95% confidence interval [95% CI] of 0.88 to 0.94), sensitivity was 0.88 (95% CI of 0.73 to 0.95), and specificity was 0.95 (95% CI of 0.84 to 1). Thus, FC is a rapid and reliable method (<2 h) to assess the effect of antifungal combinations.

Cerium, chitosan and hamamelitannin as novel biofilm inhibitors?

OBJECTIVES: The colonization of indwelling medical devices and subsequent biofilm formation represents a global challenge since it promotes the persistence of infection and contributes to antimicrobial resistance. The aim of this study was to determine the antimicrobial activity of cerium, chitosan and hamamelitannin against usual microbial colonizers and to assess their efficacy regarding biofilm formation on polyurethane (PUR)-like catheters. METHODS: The antimicrobial and anti-biofilm effect of cerium nitrate, low molecular weight chitosan (LMWC) and hamamelitannin was tested against Staphylococcus epidermidis, Staphylococcus aureus, Acinetobacter baumannii and Candida albicans strains. Biofilm formation was assessed with PUR-like catheter segments and the metabolic activity was quantified by colorimetry with a tetrazolium reduction assay. RESULTS: Cerium nitrate and LMWC inhibited the microbial growth of all microbial strains tested; hamamelitannin showed no inhibition. Regarding biofilm formation on PUR-like catheters, with subinhibitory concentrations: cerium nitrate significantly inhibited the metabolic activity of C. albicans; LMWC reduced the metabolic activity of S. epidermidis and C. albicans; and hamamelitannin decreased the metabolic activity of all tested bacteria, but not of yeasts. CONCLUSIONS: The microbicidal activity of cerium nitrate and LMWC was clearly demonstrated in this study, as was their fungistatic effect at lower concentrations. Hamamelitannin significantly reduced biofilm metabolic activity of all tested bacteria. These microbial inhibitors may play a promising role regarding different biomedical applications.

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.

A novel flow cytometric protocol for assessment of yeast cell adhesion.

Microbial adhesion is a field of recognized relevance and, as such, an impressive array of tools has been developed to understand its molecular mechanisms and ultimately for its quantification. Some of the major limitations found within these methodologies concern the incubation time, the small number of cells analyzed, and the operator's subjectivity. To overcome these aspects, we have developed a quantitative method to measure yeast cells' adhesion through flow cytometry. In this methodology, a suspension of yeast cells is mixed with green fluorescent polystyrene microspheres (uncoated or coated with host proteins). Within 2 h, an adhesion profile is obtained based on two parameters: percentage and cells-microsphere population's distribution pattern. This flow cytometry protocol represents a useful tool to quantify yeast adhesion to different substrata in a large scale, providing manifold data in a speedy and informative manner.

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.

Transcriptional profiling of azole-resistant Candida parapsilosis strains.

Herein we describe the changes in the gene expression profile of Candida parapsilosis associated with the acquisition of experimentally induced resistance to azole antifungal drugs. Three resistant strains of C. parapsilosis were obtained following prolonged in vitro exposure of a susceptible clinical isolate to constant concentrations of fluconazole, voriconazole, or posaconazole. We found that after incubation with fluconazole or voriconazole, strains became resistant to both azoles but not to posaconazole, although susceptibility to this azole decreased, whereas the strain incubated with posaconazole displayed resistance to the three azoles. The resistant strains obtained after exposure to fluconazole and to voriconazole have increased expression of the transcription factor MRR1, the major facilitator transporter MDR1, and several reductases and oxidoreductases. Interestingly, and similarly to what has been described in C. albicans, upregulation of MRR1 and MDR1 is correlated with point mutations in MRR1 in the resistant strains. The resistant strain obtained after exposure to posaconazole shows upregulation of two transcription factors (UPC2 and NDT80) and increased expression of 13 genes involved in ergosterol biosynthesis. This is the first study addressing global molecular mechanisms underlying azole resistance in C. parapsilosis; the results suggest that similarly to C. albicans, tolerance to azoles involves the activation of efflux pumps and/or increased ergosterol synthesis.

Candida krusei reservoir in a neutropaenia unit: molecular evidence of a foe?

Candida krusei has been documented as an emerging pathogen causing nosocomial outbreaks. The consecutive isolation of C. krusei strains in three patients admitted to the same hospital department within 2 months lead us to consider the possibility of an outbreak. Additionally, C. krusei isolates were collected from the room surfaces, whereas another isolate had been recovered from the blood of one patient 2 years before. HinfI DNA restriction endonuclease-based analysis of all C. krusei isolates was performed and restriction profiles were compared. Surprisingly, isolates from different patients were unrelated, whereas isolates from biological products of the same patient showed indistinguishable HinfI restriction patterns and were similar to those obtained from the surrounding environment of the respective patients. The study approach revealed the endogenous origin of the C. krusei infectious episodes observed and demonstrated that, subsequent to colonizing a patient, C. krusei can be involved in infectious episodes distant in time. The hypothesis of an outbreak was excluded, although we believe that the methodology employed in the present study represents a valuable tool for diagnostic and epidemiological surveys.

Genital candidosis in heterosexual couples.

BACKGROUND: Evidence suggests that Candida can be sexually transmitted; however, the contribution of sexual transmission to the pathogenesis of genital candidosis needs further elucidation. OBJECTIVE: The aim was to evaluate genital candidosis and its transmissibility in heterosexual couples. METHODS: Heterosexual couples were recruited among attendees of an Sexually Transmitted Diseases clinic. Specimens for yeast culture were collected from the glans penis and inner preputial layer using direct impression on CHROMagar Candida medium; vaginal exudates were collected using a cotton swab with subsequent inoculation on CHROMagar Candida medium. Mitochondrial DNA restriction analysis was performed to compare Candida isolates from both partners. RESULTS: A total of 64 couples were enrolled in the study. Frequency of sexual intercourse was significantly higher in couples where both partners yielded positive cultures and with at least one having genital candidosis (Odds ratios: 6.844; 95% CI 1.408-33.266). The same Candida species was found in both partners in 25% (16/64) of all couples but only 17.2% (11/64) were genetically similar. In total 12 of the 34 women suffering from vulvovaginal candidosis (VVC) had recurrent VVC (RVVC); two sexual partners of RVVC women (16.7%) had candida positive cultures, compared with 15 (68.2%) sexual partners of non RVVC women (Odds ratios: 0.093; 95% CI 0.016-0.544). CONCLUSIONS: Only in a few heterosexual couples a genetic similarity of Candida species recovered from both partners was found. RVVC women were more likely to have an asymptomatic candida negative sexual partner. This study suggests that male genitalia do not represent a relevant reservoir for RVVC; thus, the relevance of sexual transmission should not be emphasized.

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.