Global Epidemiology of Invasive Infections by Uncommon Candida Species: A Systematic Review.

Emerging and uncommon Candida species have been reported as an increasing cause of invasive Candida infections (ICI). We aim to systematize the global epidemiology associated with emergent uncommon Candida species responsible for invasive infections in adult patients. A systematic review (from 1 January 2001 to 28 February 2023) regarding epidemiological, clinical, and microbiological data associated to invasive Candida infections by uncommon Candida spp. were collected. In total, 1567 publications were identified, and 36 were selected according to inclusion criteria (45 cases). The chosen studies covered: C. auris (n = 21), C. haemulonii (n = 6), C. fermentati (n = 4), C. kefyr (n = 4), C. norvegensis (n = 3), C. nivariensis (n = 3), C. bracarensis (n = 1), C. duobushaemulonii (n = 1), C. blankii (n = 1), and C. khanbhai (n = 1). Over the recent years, there has been an increase in the number of invasive infections caused by uncommon Candida spp. Asia and Europe are the continents with the most reported cases. The challenges in strain identification and antifungal susceptibility interpretation were significant. The absence of clinical breakpoints for the susceptibility profile determination for uncommon Candida spp. makes interpretation and treatment options a clinical challenge. It is crucial that we focus on new and accessible microbiology techniques to make fast and accurate diagnostics and treatments.

Three-Dimensional Melted Electrowriting Drug Coating Fibers for the Prevention of Device-Associated Infections: A Pilot Study.

Medical device-related infections (DRIs), especially prevalent among critically ill patients, impose significant health and economic burdens and are mainly caused by bacteria. Severe infections often necessitate device removal when antibiotic therapy is inefficient, delaying recovery. To tackle this issue, PCL drug-eluting coated meshes were explored, and they were printed via melt electrowriting (MEW). These meshes were coated with gentamicin sulfate (GS) and tetracycline hydrochloride (TCH) and underwent FTIR analysis to confirm drug integration. Antimicrobial activity was assessed via agar diffusion assays and biofilm formation assays against bacterial strains: Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 43300, and Staphylococcus epidermidis ATCC 35984. FTIR analysis evidenced the presence of the drugs in the meshes. TCH displayed broad-spectrum antimicrobial activity against all strains, whereas GS was effective against all except S. aureus. These findings indicate the potential of cost-effective ultra-fine drug coating fibers for medical device applications, offering infection prevention during implantation. This preliminary study demonstrates the feasibility of producing drug-eluting fibers for DRI prevention through a non-toxic, fast, and cost-efficient technique, paving the way for enhanced patient care and reduced healthcare costs.

Development and Evaluation of Microencapsulated Oregano Essential Oil as an Alternative Treatment for Candida albicans Infections.

Vulvovaginal candidiasis (VVC) is characterized as a very common fungal infection that significantly affects women's health worldwide. Essential oils (EOs) are currently being evaluated as an alternative therapy. The development of efficient techniques such as micro- or nanoencapsulation for protecting and controlling release is essential to overcome the limitations of EO applications. Therefore, the aim of this study was to develop and characterize oregano EO-loaded keratin microparticles (OEO-KMPs) as a potential treatment for VVC. OEO-KMPs were produced using high-intensity ultrasonic cycles and characterized in terms of morphological and physicochemical parameters. In vitro evaluation included assessing the toxicity of the OEO-KMPs and their effect against Candida albicans using microdilution and agar diffusion, while the activity against biofilm was quantified using colony forming units (CFU). The efficacy of the OEO-KMPs in an in vivo VVC mouse model was also studied. Female BALB/c mice were intravaginally infected with C. albicans, 24 h postinfection animals were treated intravaginally with 15 µL of OEO-KMPs and 24 h later vaginal fluid was analyzed for C. albicans and Lactobacillus growth (CFU mL-1). The results showed the stability of the OEO-KMPs over time, with high encapsulation efficiency and controlled release. This nanoparticle size facilitated penetration and completely inhibited the planktonic growth of C. albicans. In addition, an in vitro application of 2.5% of the OEO-KMPs eradicated mature C. albicans biofilms while preserving Lactobacillus species. In in vivo, a single intravaginal application of OEO-KMPs induced a reduction in C. albicans growth, while maintaining Lactobacillus species. In conclusion, this therapeutic approach with OEO-KMPs is promising as a potential alternative or complementary therapy for VVC while preserving vaginal microflora.

Potential Environmental Reservoirs of Candida auris: A Systematic Review.

Candida auris, a multidrug-resistant yeast, poses significant challenges in healthcare settings worldwide. Understanding its environmental reservoirs is crucial for effective control strategies. This systematic review aimed to review the literature regarding the natural and environmental reservoirs of C. auris. Following the PRISMA guidelines, published studies until October 2023 were searched in three databases: PubMed, Web of Science, and Scopus. Information regarding the origin, sampling procedure, methods for laboratory identification, and antifungal susceptibility was collected and analyzed. Thirty-three studies published between 2016 and 2023 in 15 countries were included and analyzed. C. auris was detected in various environments, including wastewater treatment plants, hospital patient care surfaces, and natural environments such as salt marshes, sand, seawater, estuaries, apples, and dogs. Detection methods varied, with molecular techniques often used alongside culture. Susceptibility profiles revealed resistance patterns. Phylogenetic studies highlight the potential of environmental strains to influence clinical infections. Despite methodological heterogeneity, this review provides valuable information for future research and highlights the need for standardized sampling and detection protocols to mitigate C. auris transmission.

Brave New World of Artificial Intelligence: Its Use in Antimicrobial Stewardship-A Systematic Review.

Antimicrobial resistance (AMR) is a growing public health problem in the One Health dimension. Artificial intelligence (AI) is emerging in healthcare, since it is helpful to deal with large amounts of data and as a prediction tool. This systematic review explores the use of AI in antimicrobial stewardship programs (ASPs) and summarizes the predictive performance of machine learning (ML) algorithms, compared with clinical decisions, in inpatients and outpatients who need antimicrobial prescriptions. This review includes eighteen observational studies from PubMed, Scopus, and Web of Science. The exclusion criteria comprised studies conducted only in vitro, not addressing infectious diseases, or not referencing the use of AI models as predictors. Data such as study type, year of publication, number of patients, study objective, ML algorithms used, features, and predictors were extracted from the included publications. All studies concluded that ML algorithms were useful to assist antimicrobial stewardship teams in multiple tasks such as identifying inappropriate prescribing practices, choosing the appropriate antibiotic therapy, or predicting AMR. The most extracted performance metric was AUC, which ranged from 0.64 to 0.992. Despite the risks and ethical concerns that AI raises, it can play a positive and promising role in ASP.

Effect of Vapor-Phase Oregano Essential Oil on Resistant Candida Species Biofilms: Mechanisms of Action.

Vulvovaginal candidiasis (VVC) is one of the most prevalent vaginal infectious diseases. The increasing incidence of drug-resistant Candida strains and the limited therapeutic options make the discovery of effective alternative therapies fundamental. Essential oils (EOs) have been suggested as a promising alternative, and interestingly, vapor-phase essential oils (VP-EOs) present more advantages than their direct application. Thus, this study aims to evaluate the effect of oregano VP-EO (VP-OEO) on biofilms of antifungal-resistant vaginal isolates of Candida species (Candida albicans and Candida glabrata) and determine its mode of action. CFU, membrane integrity, and metabolic activity were evaluated. Furthermore, a reconstituted vaginal epithelium was used to mimic vaginal conditions and evaluate the effect of VP-OEO on Candida species infection, analyzed by DNA quantification, microscopy, and lactate dehydrogenase activity. The results revealed high VP-OEO antifungal activity. There was a significant reduction (>4 log CFU) in Candida species biofilms. Furthermore, the results show that the mechanisms of action of VP-OEO are related to membrane integrity and metabolic activity. The epithelium model confirms the effectiveness of VP-OEO. This study suggests that VP-EO can be considered a first approach for the development of an alternative form of VVC treatment. IMPORTANCE This work presents a new approach to the application of essential oils, exposure to the vapor phase, which can be considered a first approach for the development of a complementary or alternative form of vulvovaginal candidiasis (VVC) treatment. VVC is a significant infection caused by Candida species and remains a common disease that affects millions of women every year. The great difficulty in treating VVC and the extremely limited effective therapeutic options make the development of alternative treatments crucial. In this scope, this study aims to contribute to the development of effective, inexpensive, and nontoxic strategies for the prevention and treatment of this infectious disease, based on natural products. Moreover, this new approach has several advantages for women, such as lower costs, easy access, an easier mode of application, avoidance of skin contact, and, therefore, fewer negative impacts on women's health.

Biodegradable Bone Implants as a New Hope to Reduce Device-Associated Infections-A Systematic Review.

Bone fractures often require fixation devices that frequently need to be surgically removed. These temporary implants and procedures leave the patient more prone to developing medical device-associated infections, and osteomyelitis associated with trauma is a challenging complication for orthopedists. In recent years, biodegradable materials have gained great importance as temporary medical implant devices, avoiding removal surgery. The purpose of this systematic review was to revise the literature regarding the use of biodegradable bone implants in fracture healing and its impact on the reduction of implant-associated infections. The systematic review followed the PRISMA guidelines and was conducted by searching published studies regarding the in vivo use of biodegradable bone fixation implants and its antibacterial activity. From a total of 667 references, 23 studies were included based on inclusion and exclusion criteria. Biodegradable orthopedic implants of Mg-Cu, Mg-Zn, and Zn-Ag have shown antibacterial activity, especially in reducing infection burden by MRSA strains in vivo osteomyelitis models. Their ability to prevent and tackle implant-associated infections and to gradually degrade inside the body reduces the need for a second surgery for implant removal, with expectable gains regarding patients' comfort. Further in vivo studies are mandatory to evaluate the efficiency of these antibacterial biodegradable materials.

The Impact of Angiotensin-Converting Enzyme 2 (ACE2) Expression Levels in Patients with Comorbidities on COVID-19 Severity: A Comprehensive Review.

Angiotensin-Converting Enzyme 2 (ACE2) has been proved to be the main host cell receptor for the binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic. The SARS-CoV-2 spike (S) protein binds to ACE2 to initiate the process of replication. This enzyme is widely present in human organ tissues, such as the heart and lung. The pathophysiology of ACE2 in SARS-CoV-2 infection is complex and may be associated with several factors and conditions that are more severe in COVID-19 patients, such as age, male gender, and comorbidities, namely, cardiovascular diseases, chronic respiratory diseases, obesity, and diabetes. Here we present a comprehensive review that aims to correlate the levels of expression of the ACE2 in patients with comorbidities and with a poor outcome in COVID-19 disease. Significantly higher levels of expression of ACE2 were observed in myocardial and lung tissues in heart failure and COPD patients, respectively. An age-dependent increase in SARS2-CoV-2 receptors in the respiratory epithelium may be also responsible for the increased severity of COVID-19 lung disease in elderly people. Although the role of ACE2 is highlighted regarding the damage that can arise upon the SARS-CoV-2 invasion, there was no association observed between renin-angiotensin-aldosterone system (RAAS) inhibitors and the severity of COVID-19.

Species Distribution and Antifungal Susceptibility Profiles of Isolates from Women with Nonrecurrent and Recurrent Vulvovaginal Candidiasis.

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.

FKS1 mutation associated with decreased echinocandin susceptibility of Aspergillus fumigatus following anidulafungin exposure.

Invasive aspergillosis (IA) is a potentially lethal infection that affects mostly immunocompromised patients caused by Aspergillus fumigatus. Echinocandins are a second-line therapy against IA, used as a salvage therapy as well as for empirical or prophylactic therapy. Although they cause lysis of growing hyphal tips, they are considered fungistatic against molds. In vivo echinocandins resistance is uncommon; however, its wide clinical use could shortly lead to the emergence of A. fumigatus resistance. The aims of the present work was to assess the development of reduced echinocandins susceptibility phenotype by a A. fumigatus strain and to unveil the molecular mechanism underlying such phenotype. We induced in vitro cross-resistance to echinocandins following exposure of A. fumigatus to anidulafungin. Stability of the resistant phenotype was confirmed after removal of anidulafungin pressure. The FKS1 gene was partially sequenced and a E671Q mutation was found. A computational approach suggests that it can play an important role in echinocandin resistance. Given the emerging importance of this mechanism for clinical resistance in pathogenic fungi, it would be prudent to be alert to the potential evolution of this resistant mechanism in Aspergillus spp infecting patients under echinocandins therapeutics.

Candida albicans Antifungal Resistance and Tolerance in Bloodstream Infections: The Triad Yeast-Host-Antifungal.

Candida albicans represents the most frequent isolated yeast from bloodstream infections. Despite the remarkable progress in diagnostic and therapeutic approaches, these infections continue to be a critical challenge in intensive care units worldwide. The economic cost of bloodstream fungal infections and its associated mortality, especially in debilitated patients, remains unacceptably high. Candida albicans is a highly adaptable microorganism, being able to develop resistance following prolonged exposure to antifungals. Formation of biofilms, which diminish the accessibility of the antifungal, selection of spontaneous mutations that increase expression or decreased susceptibility of the target, altered chromosome abnormalities, overexpression of multidrug efflux pumps and the ability to escape host immune defenses are some of the factors that can contribute to antifungal tolerance and resistance. The knowledge of the antifungal resistance mechanisms can allow the design of alternative therapeutically options in order to modulate or revert the resistance. We have focused this review on the main factors that are involved in antifungal resistance and tolerance in patients with C. albicans bloodstream infections.

Assessing the impact of Medical Microbiology classes using active strategies on short- and long-term retention on medical students: an innovative study.

One of teachers' concerns, with students in general and medical students in particular, is to ensure as much as possible that information goes from students' short-term memories to their long-term memories. The present study focuses on knowledge retention in Medical Microbiology and assesses the effectiveness of some strategies implemented for short- and long-term retention. A pre- and post-test was used to assess student's learning. This study involved students of Porto University (test group). Test group participants were all attending the third year of the Medicine Degree Program. The results of post-test 1 were considered very positive and support the importance of these applied active activities and/or methodologies in Medical Microbiology for short-term retention. However, the results obtained in post-test 2 showed that knowledge retention after 9 months, despite substantial, decreases.

Potential Impact of Flow Cytometry Antimicrobial Susceptibility Testing on the Clinical Management of Gram-Negative Bacteremia Using the FASTinov® Kit.

Laboratory assessment of antimicrobial susceptibility is a prerequisite for adequate management of infections. The aim of this research was to evaluate the performance of the novel FASTinov® kit for antimicrobial susceptibility testing (AST) of Gram negative bacilli directly on positive blood cultures. One hundred and two positive blood cultures from patients of a Portuguese University Hospital were included. AST were performed with routine method, Vitek2, with FASTinov® kit, and with the gold standard microdilution. Bacteria directly extracted from blood cultures were used to inoculate the FASTinov® kit. Time-to-result as well as the number of patients receiving initially inappropriate therapy (and those in whom de-escalation would have been done) and length of stay (LOS) was recorded. Seventy percent of patients were over 70 years old and 18.6% were admitted in intensive care units. Regarding the isolates, 88.2% were Enterobacteriaceae, 9.8% Pseudomonas spp. and 1% Acinetobacter spp. Extended spectrum ß-lactamases producing-Enterobacteriaceae were found in 7.8% of cases and 10.8% were multi-drug resistant. Fifty-one hours was the mean of time-to-result for routine test (Vitek2) vs. 2 h response regarding Fastinov® test. The overall agreement between FASTinov® and the reference microdilution method was 98%. According to the susceptibility phenotype, 16.7% of patients received initially inappropriate therapy and the mean hospital LOS of these patients was significantly higher. FASTinov® kit revealed an excellent correlation with the AST standard method and provided much earlier results than Vitek2.

A Flow Cytometric and Computational Approaches to Carbapenems Affinity to the Different Types of Carbapenemases.

The synergy of carbapenem combinations regarding Enterobacteriaceae producing different types of carbapenemases was study through different approaches: flow cytometry and computational analysis. Ten well characterized Enterobacteriaceae (KPC, verona integron-encoded metallo-ß-lactamases -VIM and OXA-48-like enzymes) were selected for the study. The cells were incubated with a combination of ertapenem with imipenem, meropenem, or doripenem and killing kinetic curves performed with and without reinforcements of the drugs. A cephalosporin was also used in combination with ertapenem. A flow cytometric assay with DiBAC4-(3), a membrane potential dye, was developed in order to evaluate the cellular lesion after 2 h incubation. A chemical computational study was performed to understand the affinity of the different drugs to the different types of enzymes. Flow cytometric analysis and time-kill assays showed a synergic effect against KPC and OXA-48 producing-bacteria with all combinations; only ertapenem with imipenem was synergic against VIM producing-bacteria. A bactericidal effect was observed in OXA-48-like enzymes. Ceftazidime plus ertapenem was synergic against ESBL-negative KPC producing-bacteria. Ertapenem had the highest affinity for those enzymes according to chemical computational study. The synergic effect between ertapenem and others carbapenems against different carbapenemase-producing bacteria, representing a therapeutic choice, was described for the first time. Easier and faster laboratorial methods for carbapenemase characterization are urgently needed. The design of an ertapenem derivative with similar affinity to carbapenemases but exhibiting more stable bonds was demonstrated as highly desirable.

Clotrimazole Drug Resistance in Candida glabrata Clinical Isolates Correlates with Increased Expression of the Drug:H(+) Antiporters CgAqr1, CgTpo1_1, CgTpo3, and CgQdr2.

For years, antifungal drug resistance in Candida species has been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters. More recently, a few drug efflux pumps from the Drug:H(+) Antiporter (DHA) family have also been shown to play a role in this process, although to date only the Candida albicans Mdr1 transporter has been demonstrated to be relevant in the clinical acquisition of antifungal drug resistance. This work provides evidence to suggest the involvement of the C. glabrata DHA transporters CgAqr1, CgQdr2, CgTpo1_1, and CgTpo3 in the clinical acquisition of clotrimazole drug resistance. A screening for azole drug resistance in 138 C. glabrata clinical isolates, from patients attending two major Hospitals in Portugal, was performed. Based on this screening, 10 clotrimazole susceptible and 10 clotrimazole resistant isolates were selected for further analysis. The transcript levels of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 were found to be significantly up-regulated in resistant isolates when compared to the susceptible ones, with a level of correlation that was found to be similar to that of CgCDR2, an ABC gene known to be involved in the clinical acquisition of resistance. As a proof-of-concept experiment, the CgTPO3 gene was deleted in an azole resistant C. glabrata isolate, exhibiting high levels of expression of this gene. The deletion of CgTPO3 in this isolate was found to lead to decreased resistance to clotrimazole and fluconazole, and increased accumulation of azole drugs, thus suggesting the involvement of this transporter in the manifestation of azole resistance.

Rapid Flow Cytometry Test for Identification of Different Carbapenemases in Enterobacteriaceae.

A flow cytometry test was developed to identify carbapenemase production by Enterobacteriaceae and to discriminate between the different types of carbapenemases (classes A, B, and D). It is based on the detection of meropenem activity against bacteria, coupled with different carbapenemase inhibitors, which is assessed by flow cytometry. It represents a convenient, fast, and reliable approach (100% sensitivity and 100% specificity) for the detection and characterization of different carbapenemases.

Urinary Tract Infections in Kidney Transplant Patients Due to Escherichia coli and Klebsiella pneumoniae-Producing Extended-Spectrum ß-Lactamases: Risk Factors and Molecular Epidemiology.

Urinary tract infection (UTI) is a common complication after kidney transplantation, often associated to graft loss and increased healthcare costs. Kidney transplant patients (KTPs) are particularly susceptible to infection by Enterobacteriaceae-producing extended-spectrum ß-lactamases (ESBLs). A retrospective case-control study was conducted to identify independent risk factors for ESBL-producing Escherichia coli and Klebsiella pneumoniae in non-hospitalized KTPs with UTI. Forty-nine patients suffering from UTI by ESBL-producing bacteria (ESBL-P) as case group and the same number of patients with UTI by ESBL negative (ESBL-N) as control-group were compared. Clinical data, renal function parameters during UTI episodes, UTI recurrence and relapsing rate, as well as risk factors for recurrence, molecular characterization of isolates and the respective antimicrobial susceptibility profile were evaluated. Diabetes mellitus (p <0.007), previous antibiotic prophylaxis (p=0.017) or therapy (p<0.001), previous UTI (p=0.01), relapsing infection (p=0.019) and patients with delayed graft function after transplant (p=0.001) represented risk factors for infection by ESBL positive Enterobacteriaceae in KTPs. Interestingly, the period of time between data of transplantation and data of UTI was shorter in case of ESBL-P case-group (28.8 months) compared with ESBL-N control-group (50.9 months). ESBL-producing bacteria exhibited higher resistance to fluoroquinolones (p=0.002), trimethoprim-sulfamethoxazole (p<0.001) and gentamicin (p<0.001). Molecular analysis showed that blaCTX-M was the most common ESBL encoding gene (65.3%), although in 55.1% of the cases more than one ESBL gene was found. In 29.4% of K. pneumoniae isolates, three bla-genes (blaCTX-M-blaTEM-blaSHV) were simultaneously detected. Low estimated glomerular filtration rate (p=0.009) was found to be risk factor for UTI recurrence. Over 60% of recurrent UTI episodes were caused by genetically similar strains. UTI by ESBL-producing Enterobacteriaceae in KTPs represent an important clinical challenge regarding not only hospitalized patients but also concerning outpatients.

New Insights Regarding Yeast Survival following Exposure to Liposomal Amphotericin B.

In vitro resistance to amphotericin B is an extremely rare event among pathogenic yeasts. However, in vivo response is sometimes reduced, resulting in an unfavorable outcome. Such adverse outcomes might be related to subfungicidal plasma concentrations. We aimed to clarify the mechanisms of liposomal amphotericin B (AMB-L; AmBisome)-induced lesions and the mechanisms responsible for yeast cell recovery following exposure at plasma concentrations. The physiological statuses developing following exposure to AMB-L at simulated plasma concentrations (20 to 0.1 mg/liter) and at a constant concentration (3 mg/liter) were assessed in a 24-h time course assay. Time-kill experiments also were carried out under the same AMB-L treatment conditions. Our results suggest that yeast cells develop compensatory responses related to membrane polarization, metabolic activity, and reactive oxygen species (ROS) production after exposure to high plasma concentrations (20 to 5 mg/liter) during the first 6 h; in the remaining 18 h, when exposed to lower concentrations, cells reveal almost full recovery with no evidence of fungicidal activity. In contrast, whenever cells are exposed to a constant concentration above the MIC, despite initially exhibiting compensatory stress responses, soon afterwards they exhibit membrane depolarization, a decrease of metabolic activity, increasing ROS production, and lastly, programmed cell death and necrosis, resulting in succumbing to AMB-L fungicidal effects. This study may represent a step forward in the support of AMB-L use for clinical treatment of invasive fungal infections, since it demonstrates the importance of maintaining levels of AMB-L above the MIC in plasma and tissues to ensure it produces its fungicidal effects.

Ibuprofen potentiates the in vivo antifungal activity of fluconazole against Candida albicans murine infection.

Candida albicans is the most prevalent cause of fungemia worldwide. Its ability to develop resistance in patients receiving azole antifungal therapy is well documented. In a murine model of systemic infection, we show that ibuprofen potentiates fluconazole antifungal activity against a fluconazole-resistant strain, drastically reducing the fungal burden and morbidity. The therapeutic combination of fluconazole with ibuprofen may constitute a new approach for the management of antifungal therapeutics to reverse the resistance conferred by efflux pump overexpression.

Determination of chitin content in fungal cell wall: an alternative flow cytometric method.

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted GlycosylPhosphatidylInositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi.