Conventional and alternative antifungal therapies to oral candidiasis

Candida-associated denture stomatitis is the most common form of oral candidal infection, with Candida albicans being the principal etiological agent. Candida adheres directly or via an intermediary layer of plaque-forming bacteria to denture acrylic. Despite antifungal therapy to treat denture stomatitis, infection is reestablished soon after the treatment ceases. In addition, many predisposing factors have been identified as important in the development of oral candidiasis, including malnourishment, common endocrine disorders, such as diabetis mellitus, antibacterial drug therapy, corticosteroids, radiotherapy and other immunocompromised conditions, such as acquired immunodeficiency syndrome (AIDS). These often results in increased tolerance to the most commonly used antifungals. So this review suggests new therapies to oral candidiasis.82483

Molecular epidemiology and evaluation of Candida spp. persistence in the vaginal mucosa, in chronic cases of vulvovaginal candidosis

Vulvovaginal candidosis is an infection that affects most women of childbearing age; however epidemiological data on its incidence and risk of chronicity still need clarification, knowing that it varies from region to region. This study aimed to evaluate, in 93 vaginal isolates from 36 women of childbearing age with episodic (16) and/or chronic (20) vulvovaginal candidosis, virulence factors, genotypic characteristics and their relationship with chronicization of vulvovaginal candidosis, and to compare the characteristics of fungi from the two clinical groups in order to obtain differences that allow us to understand the causes of chronicity. We identified the strains phenotypically using the chromogenic medium CHROMagar and analysis of biochemical profiles. We evaluated the minimum inhibitory concentration of azole compounds (fluconazole and clotrimazole) using the in vitro microdilution method according to the CLSI standard at two different pHs (pH4.5 and pH7) for fluconazole, the ability to form biofilms using the crystal violet method according to Sherry et al, 2017, the ability to: a) form germ tube in Candida albicans strains according to the methodology described by Ellepola and Samaranayake, 2008, b) produce phospholipase using the adapted method Price et al, 1982, c) produce proteinase described by Vinodhini et al, 2016, d) cell wall hydrophobicity and e) ability to resist high pH. Adhesion to HeLa cells (human cervical cancer epithelial cell line) and resistance to phagocytosis by RAW 264.7 cells (murine macrophages) were tested in vitro, as well as evaluating their genetic similarity through the analysis of DNA profiles (molecular typing by RAPD). The antifungal activity of NaHCO3 against C. albicans ATCC 10231 and 12 clinical strains VVC was also determined using the microdilution method, biofilm formation and germ tube (hypha) formation in the presence of NaHCO3. For analysis and comparison of data from the two clinical groups, we used the graphpad prism 7 program for statistical analysis (χ2 test). Results: C. albicans was the most frequent species. When we compared the results of the determination of the minimum inhibitory concentration of the isolates from the two clinical groups, we did not find significant differences between them. We found that there were no significant differences between groups regarding the distribution of the number of biofilm-forming strains in sporadic cases of vulvovaginal candidosis or chronic vulvovaginal candidosis. In sporadic vulvovaginal candidosis (considering only strains with moderate germ tube capacity) there was a greater (p < 0.05) capacity to produce germ tubes in relation to chronic vulvovaginal candidosis. Regarding the proportion of isolates strong in producing phospholipase, no significant differences were found between sporadic and chronic isolates. The evaluation of the DNA profiles of consecutive isolates of chronic vulvovaginal candidosis revealed differences. Yeast isolates involved in sporadic infection adhered in greater proportion to HeLa cells (related to their ability to produce biofilm) (p < 0.05), while the ability to evade phagocytosis (related to high protein production) (p < 0.05) was more evident in the chronic isolates; the resistance of sporadic cases to phagocytosis related to a greater hydrophobicity of the cell wall (p < 0.05). We found that C. albicans had the ability to proliferate in broths over a wide range of pH values (7.40 to 9.77) and that the minimum inhibitory concentration of NaHCO3 against C. albicans was 12.5 mg/ml (pH 8.97). It was not possible to determine the minimum lethal concentration, which suggests that it is a fungistatic mechanism of action. C. albicans cells exposed to NaHCO3 (at concentrations at least twice the minimum inhibitory) showed a 1.5-fold reduction in the normal growth rate and a 93% reduction in hyphae-producing cells, when compared to non-exposed cells to NaHCO3. Furthermore, there was a 50% reduction in biofilm mass when C. albicans cells were exposed to four times the minimum inhibitory concentration. Conclusions: Our results show that, interestingly, one of the strategies for Candida spp persists in the host and express few virulence factors, thus triggering a lower defense reaction, and that isolates from episodic cases are strong producers of biofilms and germ tubes, which helps to adhere to Hela cells (and therefore to the vaginal epithelium), probably related in some way to the hydrophobicity of the wall. Furthermore, when in contact with macrophages these isolates persist, do not form as much biofilm or adhere as well to the epithelium, but are better resistant to phagocytosis because they produce more proteases and are more resistant to pH variations. The strains originating from chronic cases also have the ability to model virulence factors over time, which allows them to better persist in the host. We conclude that in the presence of NaHCO3 the growth of clinical and collection isolates (ATCC 10231) had, regardless of pH, reduced growth, attenuation of the formation of biofilms and hyphae, so this compound is profiled as an adjuvant for therapy with a view to controlling the chronicization of vulvovaginal candidosis.A candidose vulvovaginal é uma infeção que afeta a maioria das mulheres em idade fértil; contudo dados epidemiológicos sobre a sua incidência e risco de cronicização ainda carecem de esclarecimento, sabendo-se que varia de região para região. Este estudo visou avaliar em 93 isolados vaginais de 36 mulheres em idade fértil com candidose vulvovaginal episódica (16) e/ou crónica (20), fatores de virulência, características genotípicas e a relação destas com cronicização da candidose vulvovaginal, e comparar as características dos fungos provenientes dos dois grupos clínicos de modo a obter diferenças que permitam perceber as causas da cronicização. Identificamos as estirpes fenotipicamente, utilizando o meio cromogénico CHROMagar e análise dos perfis bioquímicos. Avaliamos a concentração mínima inibitória dos compostos azólicos (fluconazol e clotrimazol) utilizando o método de microdiluição in vitro segundo a norma CLSI em dois diferentes pHs (pH4.5 e pH7) para o fluconazol, a capacidade de: a) formar biofilmes utilizando o método de cristal violeta segundo Sherry et al, 2017, b) formar tubo germinativo nas estirpes Candida albicans segundo a metodologia descrita por Ellepola e Samaranayake, 2008, c) produzir fosfolipase utilizando o método adaptado Price et al, 1982, e) produzir proteinase descrito por Vinodhini et al, 2016 e a f) hidrofobicidade da parede celular e a capacidade de resistir a pHs altos. Adesão a células HeLa (linha de células epiteliais de câncer cervical humano) e a resistência à fagocitose por células RAW 264.7 (macrófagos murinos), foram testadas in vitro, a sua semelhança genética foi avaliada através da análise dos perfis de ADN (tipagem molecular por RAPD). Também foi determinada a atividade antifúngica de NaHCO3 contra C. albicans ATCC 10231 e 12 estirpes clínicas VVC usando o método de microdiluição, a formação de biofilme e do tubo germinativo (hifa) em presença do NaHCO3. Para comparação dos dados dos dois grupos clínicos utilizamos o programa graphpad prism 7 para análise estatística (teste do χ2). Resultados C. albicans foi a espécie mais frequente. Quando comparamos os resultados da determinação da concentração mínima inibitória dos isolados dos dois grupos clínicos, não encontramos diferenças significativas entre eles. Verificamos que não houve diferenças significativas entre os grupos, relativamente à distribuição do número de estirpes formadoras de biofilme em casos esporádicos de candidose vulvovaginal ou de candidose vulvovaginal crónica. Na candidose vulvovaginal esporádica (considerando apenas estirpes com capacidade de formar tubo germinativo moderada) verificou-se uma maior (p <0.05) capacidade de produzir tubos germinativos em relação a candidose vulvovaginal crónica. Em relação à proporção de isolados fortes em produzir fosfolipase, não foram encontradas diferenças significativas entre os isolados esporádicos e crónicos. A avaliação dos perfis de DNA de isolados consecutivos de candidose vulvovaginal cronica revelou existirem diferenças. Os isolados de levedura envolvidos na infeção esporádica aderiram em maior proporção às células HeLa (relacionada com a sua capacidade de produzir biofilme) (p <0,05), enquanto que a capacidade de evadir a fagocitose (relacionada com produção elevada de protéases) (p <0,05) foi mais evidente nos isolados crónicos; sendo a resistência dos casos esporádicos à fagocitose relacionada com uma maior hidrofobicidade da parede celular (p <0,05). Verificámos que C. albicans tinha a capacidade de proliferar em caldos numa ampla faixa de valores de pH (de 7,40 a 9,77) e que a concentração inibitória mínima de NaHCO3 contra C. albicans foi de 12,5 mg / ml (pH 8,97). Não foi possível determinar a concentração letal mínima, o que sugere tratar-se de um mecanismo de ação fungistático. As células de C. albicans expostas ao NaHCO3 (em concentrações pelo menos duas vezes a mínima inibitória), apresentaram redução de 1,5 vezes na taxa de crescimento normal e redução de 93% nas células produtoras de hifas, quando comparadas às células não expostas ao NaHCO3. Além disso, houve redução de 50% na massa do biofilme, quando as células de C. albicans foram expostas a quatro vezes mais do que a concentração mínima inibitória. Conclusões: Os nossos resultados mostram que, curiosamente, uma das estratégias para Candida spp persistirem no hospedeiro e expressarem poucos fatores de virulência, e assim desencadearem menor reação de defesa, e que os isolados de casos episódicos são fortes produtores de biofilmes e de tubos germinativos, o que ajuda a aderir a células Hela (e, portanto, ao epitélio vaginal), provavelmente relacionado de alguma forma com a hidrofobicidade da parede. Mais ainda, quando em contacto com os macrófagos estes isolados persistem, não formam tanto biofilme nem aderem tao bem ao epitélio, mas resistem melhor à fagocitose porque produzem mais protéases e são mais resistentes a variações de pH. As estirpes provenientes de casos crónicos têm também a capacidade de modelar os fatores de virulência ao longo do tempo, o que lhes permite melhor persistir no hospedeiro. Concluímos que em presença de NaHCO3 o crescimento dos isolados clínicos e de coleção (ATCC 10231) tiveram independentemente do pH, crescimento reduzido, atenuação da formação de biofilmes e hifas, pelo que se perfila este composto como um adjuvante para terapia com vista ao controle da cronicização da candidose vulvovaginal

Characterization of candida oral flora in HIV-1 infected children in the HAART era

Orientador: Maria Marluce dos Santos VilelaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: O presente estudo caracterizou a flora oral de Candida em 52 crianças infectadas pelo HIV-1 em dois períodos, definidos como período I antes da introdução de inibidores de protease no esquema de terapia antiretroviral para HIV-1 e período II após a introdução de inibidores de protease. Comparou-se as espécies de Candida identificadas nos períodos I e II. Isolados do períodos I foram identificados e as crianças em sua maioria (80%) estavam colonizadas por C. albicans. Redução no percentual de colonização por C. albicans de 80% para 52%, nos períodos I e II respectivamente, sugere mudança na colonização oral por Candida após a introdução da terapêutica com inibidores de protease HIV (IP). Destaca-se particularmente o aumento da incidência de isolados Não¿albicans (p=0.005) no período II. No período I haviam 8 crianças que estavam colonizadas por espécies Não-albicans e no período II haviam 20 crianças colonizadas com isolados Não-albicans. Investigou-se a prevalência de C. dubliniensis na família de uma das crianças que estava colonizada por esta levedura. Do total de 52 crianças 38.4% mostraram manifestação oral associada a colonização por Candida. Observou-se alta sensibilidade dos isolados aos agentes antifúngicos testados, mas 4% dos isolados exibiram resistência ao fluconazol. Documentou-se resistência cruzada entre agentes antifúngicos em isolado de Candida albicans em uma criança infectada pelo HIV-1 não previamente exposta a azoles. Um isolado de C. tropicalis mostrou baixa susceptibilidade ao fluconazol (MIC = 64 µg/ml) (1). O presente estudo revelou mudança significativa na colonização oral por Candida em crianças infectadas pelo HIV-1 sob terapia HAART (Highly Active Antiretroviral Therapy). Houve alta diversidade de espécies de Candida, com emergência de espécies Não-albicans após o uso de Inibidores de proteaseAbstract: This study characterized the Candida oral flora from 52 Brazilian HIV 1-infected children, comparing the Candida species identified in two periods before (PI) and under (PII) the introduction of the HIV Protease Inhibitor therapy. The majority (80%) of the children from the PI group were colonized by C. albicans. Children in the PII (52%) were colonized by C. albicans and 28% of them carried on mixed colonization (C. albicans and Non¿albicans isolates). Therefore when we compared the periods I and II, after the inhibitor protease usage there was an important decrease in the percentile of colonization for C. albicans of 80% to 52%, suggesting an important change of the Candida oral colonization after the HIV protease inhibitors introduction. Particularly with increase of the Non-albicans isolates incidence (p=0.005) in the period II. In PI there were 8 children that were colonized by Non-albicans species and in the PII there were 20 children colonized with Non¿albicans isolates. Rare Candida species were identified, particularly we investigated the C. dubliniensis prevalence in a HIV-infected child¿s family. Of 52 children in this study 20 (38.4%) of them showed oral lesions associated to the Candida colonization. In spite of the high susceptibility of the isolates to the antifungal agents tested in this study, 4.4% (n=2) exhibited resistance to the fluconazole. One of the isolates was C. albicans from a HIV-infected child not prior exposure to azoles, which showed antifungal cross-resistance. One C. tropicalis isolate has shown low susceptibility to fluconazol (MIC = 64 µg/ml). This study was succeeded in showing the inhibitory effect of ritonavir on a single hyphae tip growth of C. albicans. The present investigation revealed a significative change of the Candida oral colonization in Brazilian HIV-infected children under HAART, high diversity of Candida species and Non-albicans species emergence after IP usageDoutoradoPediatriaDoutor em Saude da Criança e do Adolescent

Factors influencing the presence of Candida dubliniensis and other non-albicans species in patients with oral lichen planus: a retrospective observational study

Objectives: The epidemiologic distribution of non-albicans species in the oral cavity of oral lichen planus (OLP) patients remains uncertain. Therefore, the aim of this study was to identify factors associated with the presence of C. dubliniensis and other non-albicans species. Furthermore, independent risk factors for Candida superinfection in OLP should be identified. Material and methods: Epidemiologic data and microbiological findings from 268 symptomatic OLP patients who underwent continuous oral swab culture over a 5-year period (2015-2019) were retrospectively reviewed. Candida species identification and semi-quantification were obtained by culture on CHROMagar Candida, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results: C. albicans was the most frequently isolated species (72.3%), followed by C. glabrata (7.3%), C. dubliniensis (5.8%), C. krusei and C. parapsilosis (both 2.6%). The presence of C. dubliniensis was significantly associated with tobacco smoking. Other non-albicans spp. were significantly more often detected in patients using removable dentures. Increasing age and the intake of psychotropic drugs were identified as independent risk factors of Candida superinfection in OLP. Conclusion: In OLP patients, certain local and systemic factors increase the risk of carrying potentially drug-resistant Candida species and the development of Candida superinfection of OLP lesions. Clinical relevance: Due to the frequent detection of non-albicans species in OLP, resistance or at least reduced sensitivity to azole antifungals should be expected, especially in smokers and patients using removable dentures. In the case of oral complaints, a superinfection with Candida should be considered, whereby older patients and patients taking psychotropic drugs have an increased risk for oral infection with Candida

Characteristics of Candida isolates from patients with diabetes mellitus.

The present research has investigated the molecular characterization of oral yeasts in healthy individuals from different dental clinical settings in the United Kingdom, and patients from the UK and Italy who were affected by diabetes mellitus (DM) to determine the impact of DM upon candidal infection of the mouth. In the present study of patients with DM from the UK and Italy a lower than expected incidence of oral candidal infections (7/249, 2.8%) was observed. The presence of oral yeasts and genotypic diversity of C. albicans was also not influenced by DM. There was no significant difference between the presence of oral yeasts in Italian and UK DM patients. However, more (p=0.04) C. dubliniensis isolates were found in non-DM individuals. Higher levels of Candida adhesion to fibronectin-coated paramagnetic beads were observed in isolates from DM patients. Isolates from patients with low oral yeast loads adhered significantly (p=0.0T) more than those from patients with high oral yeast loads. In general, there was no difference in proteolytic activity of isolates from DM or control patients. Candida isolates from UK DM patients had significantly greater in vitro resistance to azole antifungal agents (miconazole p<0.0001 fluconazole p=0.02 ketoconazole p=0.01) than those from Italian DM patients. In addition, C. albicans isolates from all examined patients were more susceptible to fluconazole (p=0.0008) and miconazole (p=0.01) than non-C. albicans strains. PCR fingerprinting and subsequent phylogenetic analysis revealed that C. albicans isolates from UK DM patients were the most diverse (p<0.0001) in comparison to those from Italian DM patients or from healthy subjects. Finally, it was observed that the activity of a rat IgM monoclonal anti-idiotypic antibody (mAbKlO) and a synthetic decapeptide (KP) had a significant dose- dependant fungicidal activity upon a wide spectrum of C. albicans and non-C albicans isolates from patients with and without DM

In-vitro antifungal effect of Garcinia kola and garlic (Alliums sativum) on vaginal isolates of candida

Background/Objectives:Within the last decade there has been an emergence of antifungal drug resistance. Alliums sativum and Garcinia kola seeds were tested for their anticandidal properties in comparison with fluconazole and miconazole.Methods: High Vaginal swab samples from patients with vulvovaginal candidiasis were processed and identified to the species level by germ tube method, morphology on corn meal agar and sugar fermentation reactions. Methanol and aqueous extracts of Garcinia kola and Alliums sativum, as well as fluconazole and miconazole were tested in-vitro using the agar dilution method.Results: One hundred and twenty six women with symptoms of  vulvovaginal candidiasis were sampled and Candida species were isolated from 25 of them. Candida spp. identified were C. albicans (44%), C. tropicalis (28%), C. glabrata (16%) and C. parapsilosis (12%). All species except C. glabrata were inhibited by fluconazole and miconazole, all isolates of the same species having same minimum inhibitory  concentrations (MICs). The highest MICs (25 mg/ml) with the alcoholic extracts were shown by C. albicans and C. glabrata and the lowest MICs (12.5 mg/ml) were shown by C parapsilosis and C tropicalis. All the isolates tested with Garcinia kola aqueous extract had a uniform MIC of 50 mg/ml, those tested with garlic aqueous extract had an MIC of 200 mg/ml. C. albicans and C. glabrata had MIC of 200 mg/ml of the alcoholic extract but C. tropicalis was inhibited at 25 mg/ml.Conclusion: We found that Garcinia kola and Alliums ativum have activity against the vaginal Candida species isolated thus showing promise as alternative therapy for vaginal candidiasis.Keywords: Alliums ativum, Candida spp, Garcinia kola, Minimum inhibitory concentration

Pathogenesis and treatment of oral candidosis

Oral infections caused by yeast of the genus Candida and particularly Candida albicans (oral candidoses) have been recognised throughout recorded history. However, since the 1980s a clear surge of interest and associated research into these infections have occurred. This has largely been due to an increased incidence of oral candidosis over this period, primarily because of the escalation in HIV-infection and the AIDS epidemic. In addition, changes in medical practice leading to a greater use of invasive clinical procedures and a more widespread use of immunosuppressive therapies have also contributed to the problem. Whilst oral candidosis has previously been considered to be a disease mainly of the elderly and very young, its occurrence throughout the general population is now recognised. Candida are true ‘opportunistic pathogens’ and only instigate oral infection when there is an underlying predisposing condition in the host. Treatment of these infections has continued (and in some regards continues) to be problematic because of the potential toxicity of traditional antifungal agents against host cells. The problem has been compounded by the emergence of Candida species other than C. albicans that have inherent resistance against traditional antifungals. The aim of this review is to give the reader a contemporary overview of oral candidosis, the organisms involved, and the management strategies that are currently employed or could be utilised in the future

Candida species biofilms' antifungal resistance

Candida infections (candidiasis) are the most prevalent opportunistic fungal infection on humans and, as such, a major public health problem. In recent decades, candidiasis has been associated to Candida species other than Candida albicans. Moreover, biofilms have been considered the most prevalent growth form of Candida cells and a strong causative agent of the intensification of antifungal resistance. As yet, no specific resistance factor has been identified as the sole responsible for the increased recalcitrance to antifungal agents exhibited by biofilms. Instead, biofilm antifungal resistance is a complex multifactorial phenomenon, which still remains to be fully elucidated and understood. The different mechanisms, which may be responsible for the intrinsic resistance of Candida species biofilms, include the high density of cells within the biofilm, the growth and nutrient limitation, the effects of the biofilm matrix, the presence of persister cells, the antifungal resistance gene expression and the increase of sterols on the membrane of biofilm cells. Thus, this review intends to provide information on the recent advances about Candida species biofilm antifungal resistance and its implication on intensification of the candidiasis.The authors acknowledge the Fundação para a Ciência e Tecnologia (FCT), Portugal, for supporting Maria Elisa Rodrigues (SFRH/BPD/95401/2013) and Célia F. Rodrigues (SFRH/BD/93078/2013). This study was also supported by the Programa Operacional, Fatores de competitividade—COMPETE and by national funds through FCT—Fundação para a Ciência e a Tecnologia under the scope of the projects FCT PTDC/EBB-EBI/120495/2010 and RECI/EBB-EBI/0179/2012 (FCOM-01-0124-FEDER-027462)

Diagnosis and treatment of mucosa Candida spp. infections – a review article

Candida albicans is the most common cause of fungal infections worldwide. Non-albicans Candida species play an important role in vulvovaginal candidiasis and invasive infections. Most cases of infections are endogenous. In case of patients with immune disorders this opportunistic pathogen causes both surface, systemic infections, and candidemia. Symptoms depend on the area affected. Candidiasis are treated with antimycotics; these include clotrimazole, nystatin, fluconazole, voriconazole, amphotericin B, and echinocandins. The emergence of drug resistance and the side effects of currently available antifungals are becoming a major problem in the management of Candida spp. infection