Virulence-Related Genes Expression in Planktonic Mixed Cultures of Candida albicans and Non-Albicans Candida Species.

INTRODUCTION: Candida albicans is the most common opportunistic pathogen causing fungal infections worldwide, especially in high-risk patients. Its pathogenicity is related to virulence factors gene expression, such as hyphal growth (HWP1), cell adhesion (ALS3), and protease secretion (SAP1) during infection spreading mechanisms. In recent years, an increase in non-albicans Candida infections has been reported, which may present coinfection or competitive interactions with C. albicans, potentially aggravating the patient's condition. This study aims to evaluate the expression of genes related to virulence factors of C. albicans and non-albicans Candida during planktonic stage. METHODS: C. albicans (ATCC MYA-3573) as well as with three clinical strains (C. albicans DCA53, C. tropicalis DCT6, and C. parapsilosis DCP1) isolated from blood samples, were grown in 24-well plates at 37°C for 20 h, either in monocultures or mixed cultures. Quantitative real-time polymerase chain reaction was used to evaluate the expression levels of the genes HWP1, ALS3, and SAP1 in cells collected during the planktonic stage. In addition, hyphal filamentation was observed using a Scanning Electron Microscope. RESULTS: The overexpression of HWP1 and ASL3 genes in mixed growth conditions between C. albicans and non-albicans Candida species suggests a synergistic relationship as well as an increased capacity for hyphal growth and adhesion. In contrast, C. parapsilosis versus C. tropicalis interaction shows an antagonistic relationship during mixed culture, suggesting a decreased virulence profile of C. parapsilosis during initial coinfection with C. tropicalis. CONCLUSION: The expression of HWP1, ALS3, and SAP1 genes associated with virulence factors varies under competitive conditions among species of the genus Candida during planktonic stage.

Unveiling the antifungal mechanisms of CTP, a new copper(II)-theophylline/1,10-phenanthroline complex, on drug-resistant non-albicans Candida species.

Candida species undeniably rank as the most prevalent opportunistic human fungal pathogens worldwide, with Candida albicans as the predominant representative. However, the emergence of non-albicans Candida species (NACs) has marked a significant shift, accompanied by rising incidence rates and concerning trends of antifungal resistance. The search for new strategies to combat antifungal-resistant Candida strains is of paramount importance. Recently, our research group reported the anti-Candida activity of a coordination compound containing copper(II) complexed with theophylline (theo) and 1,10-phenanthroline (phen), known as "CTP" - Cu(theo)2phen(H2O).5H2O. In the present work, we investigated the mechanisms of action of CTP against six medically relevant, antifungal-resistant NACs, including C. auris, C. glabrata, C. haemulonii, C. krusei, C. parapsilosis and C. tropicalis. CTP demonstrated significant efficacy in inhibiting mitochondrial dehydrogenases, leading to heightened intracellular reactive oxygen species production. CTP treatment resulted in substantial damage to the plasma membrane, as evidenced by the passive incorporation of propidium iodide, and induced DNA fragmentation as revealed by the TUNEL assay. Scanning electron microscopy images of post-CTP treatment NACs further illustrated profound alterations in the fungal surface morphology, including invaginations, cavitations and lysis. These surface modifications significantly impacted the ability of Candida cells to adhere to a polystyrene surface and to form robust biofilm structures. Moreover, CTP was effective in disassembling mature biofilms formed by these NACs. In conclusion, CTP represents a promising avenue for the development of novel antifungals with innovative mechanisms of action against clinically relevant NACs that are resistant to antifungals commonly used in clinical settings.

The impact of increasing non-albicans Candida trends on diagnostics in immunocompromised patients.

Invasive candidiasis (IC) represents a growing concern worldwide, with a considerable increase in non-albicans Candida (NAC) species. The study's primary goal was to determine if species identification by semi-nested PCR (sn-PCR) with primers for the five most prevalent Candida species is sufficient to deal with the current trends of Candida infections in cancer patients. Over one year, Candida isolates were collected from samples of patients with hematological and solid organ tumors in a single center. Species of Candida were identified by chromagar and multiplex sn-PCR using specific primers for Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, and the Candida parapsilosis complex. Most Candida infection episodes are caused by NAC species (70.5% of 105 isolates). Rare species (14 isolates) accounted for 13.3% of isolates and were not identified by sn-PCR using the five most common Candida species primers. More than half of these rare species caused candidemia in cancer patients (57.1%; p = 0.011). The risk factor for candidiasis was recent surgeries (p = 0.020) in adults and chemotherapy in pediatric patients (p = 0.006). Prolonged hospitalization and genitourinary tract cancer were significantly associated with invasive infections (p = 0.005 and 0.049, respectively). Recent surgery was a significant risk factor associated with C. parapsilosis and C. glabrata infections (P = 0.038 and 0.003, respectively), while C. tropicalis was significantly more common in patients with hematological malignancies (P = 0.012). Techniques with a broader identification spectrum than the major five Candida species are crucial for the optimal management of cancer patients.

Development of Echinocandin Resistance in Candida haemulonii: An Emergent, Widespread, and Opportunistic Fungal Pathogen.

Echinocandins, used for the prevention and treatment of invasive fungal infections, have led to a rise in breakthrough infections caused by resistant Candida species. Among these species, those belonging to the Candida haemulonii complex are rare multidrug-resistant (MDR) yeasts that are frequently misidentified but have emerged as significant healthcare-associated pathogens causing invasive infections. The objectives of this study were to investigate the evolutionary pathways of echinocandin resistance in C. haemulonii by identifying mutations in the FKS1 gene and evaluating the impact of resistance on fitness. After subjecting a MDR clinical isolate of C. haemulonii (named Ch4) to direct selection using increasing caspofungin concentrations, we successfully obtained an isolate (designated Ch4'r) that exhibited a high level of resistance, with MIC values exceeding 16 mg/L for all tested echinocandin drugs (caspofungin, micafungin, and anidulafungin). Sequence analysis revealed a specific mutation in the resistant Ch4'r strain, leading to an arginine-histidine amino acid substitution (R1354H), occurring at the G4061A position of the HS2 region of the FKS1 gene. Compared to the wild-type strain, Ch4'r exhibited significantly reduced growth proliferation, biofilm formation capability, and phagocytosis ratio, indicating a decrease in fitness. Transmission electron microscopy analysis revealed alterations in cell wall components, with a notable increase in cell wall thickness. The resistant strain also exhibited higher amounts (2.5-fold) of chitin, a cell wall-located molecule, compared to the wild-type strain. Furthermore, the resistant strain demonstrated attenuated virulence in the Galleria mellonella larval model. The evolved strain Ch4'r maintained its resistance profile in vivo since the treatment with either caspofungin or micafungin did not improve larval survival or reduce the fungal load. Taken together, our findings suggest that the acquisition of pan-echinocandin resistance occurred rapidly after drug exposure and was associated with a significant fitness cost in C. haemulonii. This is particularly concerning as echinocandins are often the first-line treatment option for MDR Candida species.

Systemic Infection by Non-albicans Candida Species Affects the Development of a Murine Model of Multiple Sclerosis.

Candidiasis may affect the central nervous system (CNS), and although Candida albicans is predominant, non-albicans Candida species can also be associated with CNS infections. Some studies have suggested that Candida infections could increase the odds of multiple sclerosis (MS) development. In this context, we investigated whether systemic infection by non-albicans Candida species would affect, clinically or immunologically, the severity of experimental autoimmune encephalomyelitis (EAE), which is an animal model used to study MS. For this, a strain of C. glabrata, C. krusei, and C. parapsilosis was selected and characterized using different in vitro and in vivo models. In these analysis, all the strains exhibited the ability to form biofilms, produce proteolytic enzymes, and cause systemic infections in Galleria mellonella, with C. glabrata being the most virulent species. Next, C57BL/6 mice were infected with strains of C. glabrata, C. krusei, or C. parapsilosis, and 3 days later were immunized with myelin oligodendrocyte glycoprotein to develop EAE. Mice from EAE groups previously infected with C. glabrata and C. krusei developed more severe and more prevalent paralysis, while mice from the EAE group infected with C. parapsilosis developed a disease comparable to non-infected EAE mice. Disease aggravation by C. glabrata and C. krusei strains was concomitant to increased IL-17 and IFN-γ production by splenic cells stimulated with fungi-derived antigens and with increased percentage of T lymphocytes and myeloid cells in the CNS. Analysis of interaction with BV-2 microglial cell line also revealed differences among these strains, in which C. krusei was the strongest activator of microglia concerning the expression of MHC II and CD40 and pro-inflammatory cytokine production. Altogether, these results indicated that the three non-albicans Candida strains were similarly able to reach the CNS but distinct in terms of their effect over EAE development. Whereas C. glabrata and C. Krusei aggravated the development of EAE, C. parapsilosis did not affect its severity. Disease worsening was partially associated to virulence factors in C. glabrata and to a strong activation of microglia in C. krusei infection. In conclusion, systemic infections by non-albicans Candida strains exerted influence on the experimental autoimmune encephalomyelitis in both immunological and clinical aspects, emphasizing their possible relevance in MS development.

Insights into the Multi-Azole Resistance Profile in Candida haemulonii Species Complex.

The Candida haemulonii complex (C. duobushaemulonii, C. haemulonii, and C. haemulonii var. vulnera) is composed of emerging, opportunistic human fungal pathogens able to cause invasive infections with high rates of clinical treatment failure. This fungal complex typically demonstrates resistance to first-line antifungals, including fluconazole. In the present work, we have investigated the azole resistance mechanisms expressed in Brazilian clinical isolates forming the C. haemulonii complex. Initially, 12 isolates were subjected to an antifungal susceptibility test, and azole cross-resistance was detected in almost all isolates (91.7%). In order to understand the azole resistance mechanistic basis, the efflux pump activity was assessed by rhodamine-6G. The C. haemulonii complex exhibited a significantly higher rhodamine-6G efflux than the other non-albicans Candida species tested (C. tropicalis, C. krusei, and C. lusitaneae). Notably, the efflux pump inhibitors (Phe-Arg and FK506) reversed the fluconazole and voricolazole resistance phenotypes in the C. haemulonii species complex. Expression analysis indicated that the efflux pump (ChCDR1, ChCDR2, and ChMDR1) and ERG11 genes were not modulated by either fluconazole or voriconazole treatments. Further, ERG11 gene sequencing revealed several mutations, some of which culminated in amino acid polymorphisms, as previously reported in azole-resistant Candida spp. Collectively, these data point out the relevance of drug efflux pumps in mediating azole resistance in the C. haemulonii complex, and mutations in ERG11p may contribute to this resistance profile.

Candida spp bloodstream infections in a Latin American Pediatric Oncology Reference Center: Epidemiology and associated factors.

BACKGROUND: Invasive fungal disease is a significant cause of morbidity and mortality in immunosuppressed children. The recognition of patients at risk for candidaemia is paramount to a better prognosis. OBJECTIVES: To characterize Candida spp bloodstream infections (BSI) in a reference centre for paediatric oncology and to describe the most prevalent risk factors associated with candida infections. PATIENTS/METHODS: This is a retrospective cohort study carried out with paediatric patients followed up with at the Institute of Pediatric Oncology, Brazil, who presented positive blood culture for Candida spp from January 2004 to December 2016. RESULTS: Ninety episodes of candidaemia were analysed; patients had a median age of 4.5 years, and 57.8% were males, with a diagnosis of solid tumours in 54.5% of cases. The most common Candida species were C albicans (35.6%), C parapsilosis (30.0%) and C tropicalis (16.7%). C tropicalis BSI was associated with neutropenia and skin lesions. Therapy was successful in 67.1% of the episodes. Older age and thrombocytopenia were associated with therapeutic failure. Death within 30 days occurred in 24.4% of patients; predictive factors were older age and admission to an ICU C parapsilosis candidaemia was a protective factor for death when compared to C albicans. CONCLUSION: The main species isolated were C albicans, C parapsilosis and C tropicalis. C tropicalis BSI was associated with neutropenia and skin lesions. The death rate was significant, and a worse prognosis was associated with older age, thrombocytopenia and admission to an ICU C parapsilosis infection proved to be a protective factor against mortality.

Unmasking the Amphotericin B Resistance Mechanisms in Candida haemulonii Species Complex.

The polyene amphotericin B (AMB) exerts a powerful and broad antifungal activity. AMB acts by (i) binding to ergosterol, leading to pore formation at the fungal plasma membrane with subsequent ion leakage, and (ii) inducing the intracellular accumulation of reactive oxygen species (ROS). Herein, we have deciphered the AMB resistance mechanisms in clinical isolates of Candida haemulonii complex (C. haemulonii, C. duobushaemulonii, C. haemulonii var. vulnera) in comparison to other clinically relevant non-albicans Candida species. Membrane gas chromatography-mass spectrometry analysis revealed that the vast majority of sterols were composed of ergosterol pathway intermediates, evidencing the absence of AMB target. Supporting this data, C. haemulonii species complex demonstrated poor membrane permeability after AMB treatment. Regarding the oxidative burst, AMB induced the formation of ROS in all species tested; however, this phenomenon was slightly seen in C. haemulonii complex isolates. Our results indicated that these isolates displayed altered respiratory status, as revealed by their poor growth in nonfermented carbon sources, low consumption of oxygen, and derisive mitochondrial membrane potential. The use of specific inhibitors of mitochondrial respiratory chain (complex I-IV) revealed no effects on the yeast growth, highlighting the metabolic shift to fermentative pathway in C. haemulonii strains. Also, C. haemulonii complex proved to be highly resistant to oxidative burst agents, which can be correlated with a high activity of antioxidant enzymes. Our data demonstrated primary evidence suggesting that ergosterol content, mitochondrial function, and fungal redox homeostasis are involved in AMB fungicidal effects and might explain the resistance presented in this multidrug-resistant, emergent, and opportunistic fungal complex.

New and Promising Chemotherapeutics for Emerging Infections Involving Drug-resistant Non-albicans Candida Species.

Fungal infections are a veritable public health problem worldwide. The increasing number of patient populations at risk (e.g. transplanted individuals, cancer patients, and HIV-infected people), as well as the use of antifungal agents for prophylaxis in medicine, have favored the emergence of previously rare or newly identified fungal species. Indeed, novel antifungal resistance patterns have been observed, including environmental sources and the emergence of simultaneous resistance to different antifungal classes, especially in Candida spp., which are known for the multidrug-resistance (MDR) profile. In order to circumvent this alarming scenario, the international researchers' community is engaged in discovering new, potent, and promising compounds to be used in a near future to treat resistant fungal infections in hospital settings on a global scale. In this context, many compounds with antifungal action from both natural and synthetic sources are currently under clinical development, including those that target either ergosterol or ß(1,3)-D-glucan, presenting clear evidence of pharmacologic/pharmacokinetic advantages over currently available drugs against these two well-known fungal target structures. Among these are the tetrazoles VT-1129, VT-1161, and VT-1598, the echinocandin CD101, and the glucan synthase inhibitor SCY-078. In this review, we compiled the most recent antifungal compounds that are currently in clinical trials of development and described the potential outcomes against emerging and rare Candida species, with a focus on C. auris, C. dubliniensis, C. glabrata, C. guilliermondii, C. haemulonii, and C. rugosa. In addition to possibly overcoming the limitations of currently available antifungals, new investigational chemical agents that can enhance the classic antifungal activity, thereby reversing previously resistant phenotypes, were also highlighted. While novel and increasingly MDR non-albicans Candida species continue to emerge worldwide, novel strategies for rapid identification and treatment are needed to combat these life-threatening opportunistic fungal infections.

In vitro lytic activity and antifungal susceptibility of infrequently isolated yeasts.

Non-albicans Candida species have acquired relevance in the last decades as a cause of serious disease. The virulence factors and antifungal susceptibility of these rare pathogens remain largely unrecognized. We examined a total of 50 yeast isolates corresponding to 11 different infrequently isolated yeast species for their in vitro enzymatic profile and susceptibility pattern as first-line antifungals. We found aspartyl protease activity for 100% of the isolates tested as well as variable DNAse, hemolysin, phospholipase and esterase activities. All strains had low MICs for amphotericin B and showed a variable response to fluconazole (0.125-32 µg/mL) and the echinocandins tested (0.25-> 8 µg/mL).

Identificación de las especies de Candida en las infecciones sanguíneas / Characterization of Candida species in blood stream infections

Introducción: La candidemia se convirtió en una infección importante del torrente sanguíneo que se asocia frecuentemente con índices elevados de mortalidad y morbilidad. Las especies de Candida generan del 70% al 80% de las infecciones micóticas invasivas del torrente sanguíneo y son la cuarta causa más frecuente de infecciones hospitalarias del torrente sanguíneo. La identificación de las especies de Candida es importante, ya que las especies no albicans son cada vez más numerosas y resistentes a las drogas antimicóticas. El objetivo del estudio fue aislar e identificar diferentes especies de Candida asociadas con candidemia y analizar su patrón de susceptibilidad a los antimicóticos. Materiales y métodos: Los pacientes con sospecha de infecciones del torrente sanguíneo (ITS) fueron reclutados durante un período de un año para el estudio prospectivo. Se analizaron las características demográficas, la duración de la internación y los factores de riesgo asociados y la evolución clínica. El análisis de las muestras de sangre tuvo lugar mediante el sistema automatizado BacTAlert. La identificación y la susceptibilidad antimicótica de las levaduras fueron realizadas mediante el uso de dispositivo VITEK-2. Resultados: Las especies de Candida fueron aisladas en 30 de los 3146 cultivos recibidos (0.9%). La mayoría de los casos de candidemia tuvieron lugar en hombres (66%). Los factores de riesgo más frecuentes fueron el uso de antibióticos de amplio espectro, la vía central y la ventilación mecánica. Entre las levaduras aisladas, las especies de Candida no albicans fueron predominantes (60%), en comparación con la especie C. albicans (40%). La especie albicans presentó una susceptibilidad del 100% a los azoles y la anfotericina, en tanto que las especies no albicans fueron resistentes. De los 30 pacientes mencionados, 5 fallecieron. Conclusión: La prevalencia de Candida no albicans fue mayor en comparación con la prevalencia de Candida albicans. Las especies no albicans fueron más resistentes a los antimicóticos. En consecuencia, los pacientes internados deberían ser evaluados para identificar la candidemia.

Introduction: Candidemia has become an important bloodstream infection that is frequently associated with high rates of mortality and morbidity. Candida species account for 70-80% of invasive bloodstream fungal infections and represent the fourth most common nosocomial bloodstream infections. The identification of Candida species is important as the number of non albicans Candida species is increasing and they are becoming more resistant to antifungal drugs. The aim of the study was to isolate and identify various Candida species associated with candidemia and to study their antifungal susceptibility pattern. Materials and methods: Patients suspected of having BSI were enrolled on a one-year prospective study. Patient's demographic details, duration of hospital stay, associated risk factors and outcome were studied. Blood samples were analyzed by BacTAlert automated system. Identification and antifungal susceptibility testing of yeasts was done using VITEK-2 compact system. Results: Of 3146 blood cultures received, Candida species were isolated in 30 samples (0.9%). The majority of candidemia cases were in males (66%). The most common risk factors were use of broad spectrum antibiotics, central line and mechanical ventilation. Among the yeast isolates, non albicans Candida species were predominant (60%) compared to C. albicans (40%). Candida albicans showed 100% susceptibility to azoles and amphotericin whereas non albicans Candida species showed resistance. Of these 30 patients, 5 patients died. Conclusion: Prevalence of non albicans Candida was greater than C. albicans and cases were more resistant to antifungal drugs. Therefore surveillance for candidemia should be carried out in hospitalized patients.

Peritonitis terciaria por Candida famata: infección por una levadura atípica / Tertiary peritonitis due to Candida famata: Infection by an atypical yeast

ResumenLa Candida famata es una levadura halotolerante, sobreproductora de vitamina B2, asociada infrecuentemente a infecciones en seres humanos, cuyo uso es común en procesos industriales. En nuestro servicio, se había detectado un único caso en el contexto de mediastinitis con un desenlace mortal. Se carecía de experiencia en cuanto al manejo de la infección por este agente. Posterior a una exhaustiva revisión bibliográfica, se demuestra la ausencia de reportes de infecciones por este agente en el contexto de la peritonitis terciaria. Se reporta el caso de un paciente de 37 años de edad que sufrió una herida de arma blanca en abdomen, y que después de múltiples complicaciones asociadas a este evento, desarrolló una peritonitis terciaria por C. famata; fue tratado satisfactoriamente con caspofungina y un abdomen abierto con terapia de presión negativa e instilación. Se espera que esta experiencia sirva de referente al detectar algún paciente con esta condición en el futuro.

AbstractCandida famata is a halotoleran, vitamin B2 overproducing yeast that is rarely associated with infections in humans, but is commonly utilized in industrial processes. In our service, it had been detected only once before in a patient with mediastinitis. The outcome of that infection was fatal. Not only were we unfamiliar with the management of infection by this agent, but after performing a thorough literary review, we were unable to find published reports of infections by this pathogen as the cause of tertiary peritonitis. We report the case of a 37 year old male who after suffering a stab wound to his abdomen and having multiple complications associated with this event, developed tertiary peritonitis by C. famata, that was treated with casponfungin and an open abdomen utilizing negative pressure wound therapy with instillation which yielded a satisfactory response to the therapy. This report may be of use in the event that clinicians are confronted with this condition in the future.

Candida glabrata among Candida spp. from environmental health practitioners of a Brazilian Hospital

Abstract The incidence of the species Candida albicans and non-albicans Candida was evaluated in a Brazilian Tertiary Hospital from the environment and health practitioners. In a 12-month period we had a total positivity of 19.65% of Candida spp. The most recurring non-albicans Candida species was C. glabrata (37.62%), generally considered a species of low virulence, but with a higher mortality rate than C. albicans. Subsequently, C. parapsilosis (25.74%) and C. tropicalis (16.86%) were the second and third most commonly isolated species. Considering the total samples collected from the emergency room and from the inpatient and the pediatric sector, 19.10% were positive for Candida spp., with the predominance of non-albicans Candida species (89.42%). The high percentage of positivity occurred in the hands (24.32%) and the lab coats (21.88%) of the health care assistants. No sample of C. albicans presented a profile of resistance to the drugs. All the non-albicans Candida species presented a decreased susceptibility to miconazole and itraconazole, but they were susceptible to nystatin. Most of the isolates were susceptible to fluconazole and amphotericin B. As expected, a high resistance rate was observed in C. glabrata and C. krusei, which are intrinsically less susceptible to this antifungal agent. The contamination of environmental surfaces by Candida spp. through hand touching may facilitate the occurrence of Candida infections predominantly in immunocompromised patients. In addition to that, the antifungal agents used should be carefully evaluated considering local epidemiologic trends in Candida spp. infections, so that therapeutic choices may be better guided.

Reversal of fluconazole resistance induced by a synergistic effect with Acca sellowiana in Candida glabrata strains.

CONTEXT: The increased incidence of non-albicans Candida (NAC) resistant to fluconazole (FLZ) makes it necessary to use new therapeutic alternatives. Acca sellowiana (O.berg) Burret (Myrtaceae) is a guava with several proven biological activities. The interaction with fluconazole can be a feasible alternative to overcome this resistance. OBJECTIVE: This study evaluates the in vitro antifungal activity of fractions obtained from the lyophilized aqueous extract of the leaves of A. sellowiana against resistant strains of NAC. MATERIALS AND METHODS: The antifungal activity of the fractions was evaluated at 500 µg/mL by microdilution method. Checkerboard assay was performed to determine the effect of the combination of the F2 fraction and antifungal at concentrations: MIC/4, MIC/2, MIC, MIC × 2 and MIC × 4. RESULTS: Candida glabrata showed the lowest MIC values (500-3.90 µg/mL) and the F2 active fraction was the most effective. The association of F2 with FLZ showed a strong synergistic effect (FICI ≤ 0.5) against 100% of C. glabrata resistant isolates. Moreover, the F2 active fraction has demonstrated that probably acts in the cell wall of these yeasts. There was no observed acute dermal toxicity of lyophilized aqueous extract of leaves of A. sellowiana on pig ear skin cells. DISCUSSION AND CONCLUSION: The interaction between substances present in the F2 active fraction is possibly responsible for the antifungal activity presented by this fraction. This study is unprecedented and suggests that the combination of F2 active fraction and FLZ might be used as an alternative treatment for mucocutaneus infections caused by C. glabrata resistant.

Candida glabrata among Candida spp. from environmental health practitioners of a Brazilian Hospital

The incidence of the species Candida albicans and non-albicans Candida was evaluated in a Brazilian Tertiary Hospital from the environment and health practitioners. In a 12-month period we had a total positivity of 19.65% of Candida spp. The most recurring non-albicans Candida species was C. glabrata (37.62%), generally considered a species of low virulence, but with a higher mortality rate than C. albicans. Subsequently, C. parapsilosis (25.74%) and C. tropicalis (16.86%) were the second and third most commonly isolated species. Considering the total samples collected from the emergency room and from the inpatient and the pediatric sector, 19.10% were positive for Candida spp., with the predominance of non-albicans Candida species (89.42%). The high percentage of positivity occurred in the hands (24.32%) and the lab coats (21.88%) of the health care assistants. No sample of C. albicans presented a profile of resistance to the drugs. All the non-albicans Candida species presented a decreased susceptibility to miconazole and itraconazole, but they were susceptible to nystatin. Most of the isolates were susceptible to fluconazole and amphotericin B. As expected, a high resistance rate was observed in C. glabrata and C. krusei, which are intrinsically less susceptible to this antifungal agent. The contamination of environmental surfaces by Candida spp. through hand touching may facilitate the occurrence of Candida infections predominantly in immunocompromised patients. In addition to that, the antifungal agents used should be carefully evaluated considering local epidemiologic trends in Candida spp. infections, so that therapeutic choices may be better guided.(AU)

Prevalence of fungemia in a tertiary hospital: Analysis of the last decade / Prevalência de fungemia em um hospital terciário: análise da última década

Summary Introduction: The prevalence of nosocomial fungemia has increased worldwide, and mortality caused by this disease is high. Objective: To assess progress in the last decade, and the prevalence and profile of fungal agents isolated in blood cultures performed in a tertiary university hospital. Method: All the results of blood cultures processed at Hospital das Clínicas, Universidade Federal de Minas Gerais (HC-UFMG), in the time intervals 2001-2003 and 2011-2013 were analyzed retrospectively. For each three-year period, the number of collected blood cultures, the overall positivity rate and the percentage of fungemia were recorded. In addition, all identified fungal species were cataloged. All blood samples were incubated in the BacT/ALERT® (bioMérieux) automation system. Results: In 2001-2003, 34,822 samples were evaluated, with 5,510 (15.8%) positive results. In 2011-2013, the number of blood cultures processed increased to 55,052 samples, with 4,873 (8.9%) positive results. There was an increase in the number of positive cultures for fungi in the analyzed period (2001-2003: 4.16%; 2011-2013: 5.95%; p<0.001). Among the agents, candidemias were predominant, especially those caused by non-albicans Candida species (2001-2003: 57.64%; 2011-2013: 65.17%; p<0.05). There was also an increase in fungemia caused by other genera (2001-2003: 2.62%; 2011-2013: 4.48%; p<0.01). Conclusion: There was an increase in the prevalence of fungemia in the last decade at HC-UFMG. Although candidemias have been responsible for most of the cases, there has been an increase in fungemias caused by other species.

Resumo Introdução: a prevalência de fungemia hospitalar tem aumentado em todo o mundo e a mortalidade por essa afecção é elevada. Objetivo: avaliar a evolução, na última década, da prevalência e do perfil dos agentes fúngicos isolados em hemoculturas realizadas em um hospital universitário terciário. Método: foram analisados retrospectivamente todos os resultados de hemocultura processados no Hospital das Clínicas da Universidade Federal de Minas Gerais (HC-UFMG), entre os períodos de 2001-2003 e de 2011-2013. Para cada triênio foram registrados o número de hemoculturas coletadas, o percentual de positividade geral e o percentual de fungemia. Também foram catalogadas todas as espécies fúngicas identificadas. Todas as amostras sanguíneas foram incubadas no sistema de automação BacT/ALERT® (bioMérieux). Resultados: entre 2001-2003, foram avaliadas 34.822 amostras, sendo 5.510 (15,8%) positivas. Entre 2011-2013, o número de hemoculturas processadas aumentou para 55.052 amostras, sendo 4.873 (8,9%) positivas. Observou-se um aumento do número de culturas positivas para fungos no período analisado (2001-2003: 4,16%; 2011-2013: 5,95%; p<0,001). Dentre os agentes, as candidemias foram predominantes, principalmente por espécies de Candida não albicans (2001-2003: 57,64%; 2011-2013: 65,17%; p<0,05). Houve também aumento da fungemia por outros gêneros (2001-2003: 2,62%; 2011-2013: 4,48%; p<0,01). Conclusão: houve aumento da prevalência de fungemia na última década no HC-UFMG. Embora as candidemias tenham sido responsáveis pela maioria dos casos, houve aumento de fungemias causadas por outras espécies.

Candida glabrata among Candida spp. from environmental health practitioners of a Brazilian Hospital.

The incidence of the species Candida albicans and non-albicans Candida was evaluated in a Brazilian Tertiary Hospital from the environment and health practitioners. In a 12-month period we had a total positivity of 19.65% of Candida spp. The most recurring non-albicans Candida species was C. glabrata (37.62%), generally considered a species of low virulence, but with a higher mortality rate than C. albicans. Subsequently, C. parapsilosis (25.74%) and C. tropicalis (16.86%) were the second and third most commonly isolated species. Considering the total samples collected from the emergency room and from the inpatient and the pediatric sector, 19.10% were positive for Candida spp., with the predominance of non-albicans Candida species (89.42%). The high percentage of positivity occurred in the hands (24.32%) and the lab coats (21.88%) of the health care assistants. No sample of C. albicans presented a profile of resistance to the drugs. All the non-albicans Candida species presented a decreased susceptibility to miconazole and itraconazole, but they were susceptible to nystatin. Most of the isolates were susceptible to fluconazole and amphotericin B. As expected, a high resistance rate was observed in C. glabrata and C. krusei, which are intrinsically less susceptible to this antifungal agent. The contamination of environmental surfaces by Candida spp. through hand touching may facilitate the occurrence of Candida infections predominantly in immunocompromised patients. In addition to that, the antifungal agents used should be carefully evaluated considering local epidemiologic trends in Candida spp. infections, so that therapeutic choices may be better guided.

Ação antifúngica da lactoferrina frente Candida non-albicans isoladas da cavidade oral de crianças infectadas pelo HIV / Antifungal action of lactoferrin against Candida non-albicans isolated from the oral cavity of HIV-infected children

A lactoferrina é uma glicoproteína de ligação ao ferro, que está presente na saliva, no leite, e em outras secreções exócrinas. Essa proteína tem inúmeras funções biológicas, incluindo efeitos antifúngicos e antibacterianos, além de imunomoduladores. O objetivo do presente estudo foi avaliar, in vitro, pela primeira vez, a atividade antifúngica da lactoferrina contra cepas de Candida nãoalbicans isoladas da cavidade oral de crianças infectadas pelo HIV e crianças saudáveis. Além disso, mensurar a degradação da lactoferrina por essas cepas através de SDS-PAGE (análise eletroforética em gel de poliacrilamida) e determinar, in vitro, a concentração mínima inibitória de lactoferrina capaz de matar 50% das células de Candida não-albicans. Cepas de Candida spp. foram obtidas através da coleta de saliva de 70 crianças infectadas pelo HIV e 50 crianças sem evidências de imunossupressão, com idade de 3 a 13 anos (ALVES, 2014). As diferentes espécies de Candida foram identificadas através de assimilação e fermentação de açúcar (API 20C, Biomérieux, França). Para o ensaio, in vitro, de morte de celular na presença de lactoferrina, 24 isolados de Candida, entre elas parapsilosis, tropicalis, krusei, guillermondi e dubliniensis, foram selecionados. Para a realização da análise por SDS-PAGE, cepas de todas as espécies consideradas resistentes nos ensaios de morte celular, foram incluídas. O porcentual de morte celular (%) de Candida não- albicans com a adição de 100 µg/ml de lactoferrina variou de 3,1% (C. dubliniensis) a 88,1% (C. tropicalis) no grupo HIV, e de 14,1% a 30,37% no grupo não-HIV (C . parapsilosis). Não houve correlação entre a densidade celular e o percentual de morte celular. C. dubliniensis foi a espécie mais resistente a lactoferrina e o porcentual de morte celular de C. parapsilosis foi significativamente maior em comparação com C. krusei na concentração de 1X104 células/ml (p = 0,033). Todos os isolados foram capazes de degradar a lactoferrina na concentração 1x108 células/ml, especialmente C. parapsilosis, pelo grupo HIV. Além disso, a lactoferrina na concentração de 500 µg/ml foi capaz de matar mais de 50% das células apenas dos isolados de C. tropicalis e C. guilliermondii. Concluiu-se que a lactoferrina tem atividade antifúngica contra Candida não-albicans de crianças infectadas pelo HIV, mas algumas espécies apresentam alguma resistência a esta proteína. Além disso, todas as Candida spp. foram capazes de degradar a lactoferrina quando estavam em concentração de 1X108 cels/ml, e a lactoferrina na concentração de 500µl/ml foi capaz de matar mais que 50% das células de C. tropicalis e C. guillermondii

Lactoferrin is an iron-binding glycoprotein, which is present in saliva, milk and other exocrine secretions. This protein has a number of biological functions, including antifungical, antimicrobial and immunomodulatory effects. The aim of this study was to evaluate, for the first time, the antifungal activity of lactoferrin against isolates of Candida spp. from the oral cavity of HIV-infected children and children with no clinical evidence of immunosuppression. Furthermore, measure, in vitro, the degradation of lactoferrin by Candida spp. through SDS-PAGE (electrophoretic analysis on polyacrylamide gel) and determine, in vitro, the minimum inhibitory concentration of lactoferrin able to kill 50% of cells of Candida non-albicans. Strains of Candida spp. were obtained through saliva collect of 70 HIV-infected children and 50 children without evidence of immunosuppression ageing between 3 to13 years old (ALVES, 2014). All Candida species were identified by sugar assimilation and fermentation (API 20C, Biomerieux, France). For the in vitro study, death of lactoferrin of 24 Candida isolates, including parapsilosis, tropicalis, krusei, guillermondii, and dubliniensis, were selected. To perform the analysis by SDS-PAGE, strains of all available species considered resistants, which were observed at lactoferrin death`s study, were included. The cell death percentage (%) of non-albicans Candida by addition of 100 µg of lactoferrin range from 3.1% (C. dublinienes) to 88.1% (C. tropicalis) in HIV group, and 14.1% to 30.37% in N-HIV (C. parapsilosis), but there was no correlation between cell density and death %. C. dubliniensis was the most resistant specie to lactoferrin and the cell death % of C. parapsilosis was significant higher comparing to C. krusei at 1X104 cells/ml (p=0.033). All the isolates were able to degrade lactoferrin at 108 cells/ml, mainly C. parapsilosis from HIV. Furthermore, lactoferrin at 500 µg/ml concentration was able to kill more than 50% of the cells only for C. tropicalis and C. guillermondii isolates. It was concluded that lactoferrin has antifungal activity against non-albicans Candida from HIV children, but some species present some resistance to this protein. Furthermore, all Candida spp. were able to degrade lactoferrin when was at concentration of 1x108 cells / ml, also, lactoferrin at concentration 500µl / ml was able to kill more than 50% of C. tropicalis and C. guillermondii cells.

Potencial cariogênico de Candida não-albicans isoladas do biofilme dentário de crianças infectadas pelo HIV / Cariogenic potential of Candida non-albicans isolated from the dental biofilm of HIV-infected children

O objetivo deste estudo foi analisar, in vitro, o padrão de desmineralização do esmalte bovino exposto a C. não-albicans isoladas de biofilme dental de crianças infectadas pelo HIV, em comparação com C. albicans (n = 2) e Streptococcus (S) mutans (ATCC). Também foram avaliadas as formações de biofilme e produção de L-lactato por estas espécies não-albicans. Cento e oitenta blocos de esmalte foram divididos em 6 grupos com biofilme formado por: C. glabrata; C. parapsilosis; C. tropicalis; C. albicans; S. mutans e controle negativo (meio sem biofilme). Três blocos de esmalte de cada grupo foram retirados nos dias 3, 5, 8, 15 e 28 após a formação de biofilme para avaliar a microdureza superficial do esmalte final (SMH). Além disso, a viabilidade das células do biofilme e o L-lactato produzidos por estas células foi medida por redução de XTT e enzimaticamente, respectivamente. Análise microscópica de varrimento laser confocal (CLS) foi utilizado para avaliar a topografia de cada isolado de biofilme de C. não-albicans. Os resultados da análise de SMH foram submetidos a testes de Mann-Whitney de Kruskall-Wallis e com um nível de significância de 95% (p <0,05), enquanto que os dados de viabilidade do biofilme, L-lactato e análise CLS foram avaliadas de forma descritiva. Todos os grupos mostraram um aumento na perda de SMH, mas apenas os grupos expostos a biofilmes de S. mutans e C. albicans foram estatisticamente significativas ao longo do tempo. Ao comparar as espécies C. não-albicans entre si, C. tropicalis apresentaram maior perda% no 5 º dia em comparação com C. parapsilosis e no 8 º dia em comparação com C. glabrata. Não foi observada diferença estatística entre C. parapsilosis e C. glabrata. Todos os isolados de C. não-albicans mostraram o mesmo perfil de formação de biofilme. A produção de L-lactato foi maior em C. tropicalis seguido por C. glabrata e C. parapsilosis. No entanto, a análise mostrou CLS C. parapsilosis biofilme com mais células viáveis e com a matriz extracelular mais. Embora com menor intensidade em comparação com C. albicans e S. mutans, os isolados de Candida não-albicans do biofilme dental de crianças infectadas pelo HIV, foram capazes de causar a desmineralização do esmalte bovino. (AU)

This study aimed to analyzed , in vitro, the demineralization pattern of bovine enamel exposed to biofilm of C. non-albicans isolated from dental biofilm of HIV infected children, comparing to C. albicans (n=2) and Streptococcus (S) mutans (ATCC).We also evaluated the biofilm formations and the production of L-lactate by these nonalbicans species. A hundred eigthy enamel blocks randomly assigned to 6 groups with biofilm formed by: C. glabrata; C. parapsilosis; C. tropicalis; C. albicans; S. mutans and negative control (medium without biofilm). Three enamel blocks from each group were removed on days 3, 5, 8, 15 and 28 after biofilm formation to evaluate the final enamel surface microhardness (SMH). In addition, the cells viability of the biofilm and the L-lactate produced by these cells were measured by XTT reduction and enzymatically, respectively. Confocal laser scanning microscopic (CLS) analysis was used to evaluate the biofilm architecture of each C. non-albicans isolate. The results from SMH analysis were subjected to Kruskall-Wallis and MannWhitney tests with a 95% significance level (p<0.05), while the data from biofilm viability, L-lactate and CLS analysis were evaluated descriptively. All groups showed an increase in loss of SMH but only the groups exposed to biofilms from S. mutans and C. albicans were statistically significant over time. When comparing the species C. non-albicans among themselves, C. tropicalis showed a greater % loss on the 5th day compared with C. parapsilosis and at the 8th day compared to c. glabrata. No statistical difference betwwen C. parapsilosis and C. glabrata was observed. All isolates from C. non-albicans showed the same profile of biofilm formation. The Llactate production was higher in C. tropicalis followed by C. glabrata and C. parapsilosis. However, the CLS analysis showed C. parapsilosis biofilm with more viable cells and with more extracellular matrix. Although with less intensity comparing to C. albicans and S. mutans, isolates of Candida non-albicans from HIV infected children dental biofilm, were able to cause demineralization of bovine enamel. (AU)