The postantifungal effect of nystatin and its impact on adhesion attributes of oral Candida dubliniensis isolates

The postantifungal effect (PAFE) has an impact on candidal pathogenicity. However, there is no information on either the PAFE or its impact on adhesion traits of oral Candida dubliniensis isolates. Oral candidosis can be treated topically with nystatin. Adhesion to buccal epithelial cells (BEC), germ tube (GT) formation and relative cell surface hydrophobicity (CSH) are all colonisation attributes of candidal pathogenicity. Hence, the main objective of this study was to investigate the in vitro PAFE on 20 C. dubliniensis isolates following exposure to nystatin. In addition, the impact of nystatin‐induced PAFE on adhesion to BEC, GT formation and relative CSH of C. dubliniensis isolates were also evaluated. After determining the minimum inhibitory concentration (MIC) of nystatin, C. dubliniensis isolates were exposed to sublethal concentrations of nystatin for 1 h. Following this exposure, the drug was removed and PAFE, adhesion to BEC, GT formation and relative CSH were determined by a previously described turbidometric method, adhesion assay, germ tube induction assay and biphasic aqueous‐hydrocarbon assay respectively. MIC (μg/ml) of C. dubliniensis isolates to nystatin ranged from 0.09 to 0.78. The nystatin‐induced mean PAFE (hours) on C. dubliniensis isolates was 2.17. Compared with the controls, exposure to nystatin suppressed the ability of C. dubliniensis isolates to adhere BEC, GT formation and relative CSH by a mean percentage reduction of 74.45% (P < 0.0001), 95.92% (P < 0.0001) and 34.81 (P < 0.05) respectively. Hence, brief exposure of C. dubliniensis isolates to nystatin would continue to wield an antifungal effect by suppressing growth as well as its adhesion attributes.     

Cell surface hydrophobicity of oral Candida dubliniensis isolates following limited exposure to sub‐therapeutic concentrations of chlorhexidine gluconate

Candidal adhesion has been implicated as the initial step in the pathogenesis of oral candidiasis and cell surface hydrophobicity (CSH) has been implicated in adhesion to mucosal surfaces. Candida dubliniensis is an opportunistic pathogen associated with recurrent oral candidiasis. Chlorhexidine gluconate is by far the commonest antiseptic mouth wash prescribed in dentistry. At dosage intervals the intraoral concentration of this antiseptic fluctuates considerably and reaches sub‐therapeutic levels due to the dynamics of the oral cavity. Hence, the organisms undergo only a limited exposure to the antiseptic during treatment. The impact of this antiseptic following such exposure on CSH of C. dubliniensis isolates has not been investigated. Hence, the main objective of this study was to investigate the effect of brief exposure to sub‐therapeutic concentrations of chlorhexidine gluconate on the CSH of C. dubliniensis isolates. Twelve oral isolates of C. dubliniensis were briefly exposed to three sub‐therapeutic concentrations of 0.005%, 0.0025% and 0.00125% chlorhexidine gluconate for 30 min. Following subsequent removal of the drug, the CSH of the isolates was determined by a biphasic aqueous‐hydrocarbon assay. Compared with the controls, exposure to 0.005% and 0.0025% chlorhexidine gluconate suppressed the relative CSH of the total sample tested by 44.49% (P < 0.001) and 21.82% (P < 0.018), respectively, with all isolates being significantly affected. Although exposure to 0.00125% of chlorhexidine gluconate did not elicit a significant suppression on the total sample tested (7.01%; P > 0.05), four isolates of the group were significantly affected. These findings imply that exposure to sub‐therapeutic concentrations of chlorhexidine gluconate may suppress CSH of C. dublinienis isolates, thereby reducing its pathogenicity and highlights further the pharmacodynamics of chlorhexidine gluconate.     

Changes in germ tube formation and cell‐surface hydrophobicity of oral Candida dubliniensis isolates following brief exposure to sub‐cidal concentrations of polyene and azole antifungal agents

Adherence of Candida has been implicated as the initial process in the pathogenesis of oral candidosis. Candidal germ tubes and its relative cell‐surface hydrophobicity (CSH) are contributory attributes. Candida dubliniensis is currently documented as an opportunistic pathogen allied with recurrent oral candidosis. Oral candidosis can be treated with polyene and azole antifungals such as amphotericin B, ketoconazole and fluconazole. However, the intraoral concentration of these drugs fluctuates and becomes sub‐therapeutic because of the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intraorally, the pathogenic yeast may undergo a brief exposure to antifungal drugs. The objective of this study was to investigate the effect of brief exposure to sub‐lethal concentrations of these antifungals on the germ tube formation and CSH of C. dubliniensis. After determining the minimum inhibitory concentration of the drugs, 20 oral isolates of C. dubliniensis were exposed to sub‐lethal concentrations of these antifungals for 1 h. Following this brief exposure, the drugs were removed, and following subsequent incubation in a germ tube inducing medium and exposure to bi‐phasic hydrocarbon assay, the germ tube formation and CSH of these isolates was quantified respectively. Compared with controls, exposure to amphotericin B almost completely suppressed the ability to form germ tubes with a mean percentage reduction of 95.91% (P < 0.0001), whereas ketoconazole and fluconazole also significantly inhibited germ tube formation but to a lesser degree with a mean percentage reduction of 18.73% and 12.01% respectively (P < 0.05). Compared with controls, exposure to amphotericin B and ketoconazole elicited a significant suppression on CSH with a mean percentage reduction of 33.09% and 21.42%, respectively (P < 0.001), whereas exposure to fluconazole did not elicit a significant suppression on CSH (9.21%; P > 0.05). In clinical terms it appears that, even a short exposure to sub‐lethal concentrations of these drugs, a situation all too familiar in the oral environment, would continue to exert an antifungal effect by suppressing the pathogenic potency of C. dubliniensis.     

The postantifungal effect and phospholipase production of oral Candida albicans from smokers,diabetics, asthmatics,denture wearers and healthy individuals following brief exposure to subtherapeutic concentrations of chlorhexidine gluconate

Candida albicans is the major aetiological agent of oral candidosis and one of its important virulent factors is the production of extracellular phospholipases, which can be modulated by subtherapeutic concentrations of antifungal agents thus decreasing their pathogenicity. Hence, considering that chlorhexidine gluconate (CG) is a common antimicrobial mouthwash used in dentistry and that its concentration in the mouth reaches subtherapeutic levels during dosage intervals due to the diluent effect of saliva and cleansing effect of the oral musculature, the postantifungal effect (PAFE) and the phospholipase production of oral C. albicans following brief exposure to subtherapeutic concentrations of CG was studied. Fifty C. albicans planktonic oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to three subtherapeutic concentrations of CG (0.005%, 0.0025% and 0.00125%) for 1 h. Isolates unexposed to CG was the control group. Thereafter the antiseptic was removed and the PAFE and phospholipase production was determined by a turbidometric method and a plate assay using an egg yolk agar medium respectively. Mean PAFE (hours) of 50 oral isolates of C. albicans following 1‐h exposure to 0.005%, 0.0025% and 0.00125% CG was 6.97, 1.85 and 0.62 respectively. The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 21.68, 18.20 and 14.04% following exposure to 0.005%, 0.0025% and 0.00125% CG respectively. Brief exposure of C. albicans isolates to subtherapeutic concentrations of CG would wield an antifungal effect by suppressing growth and phospholipase production, thereby quelling its pathogenicity.     

In vitro efficacy of amphotericin B, 5‐fluorocytosine,fluconazole, voriconazole and posaconazole against Candida dubliniensis isolates using time‐kill methodology

Candida dubliniensis is a recently described yeast that causes infections in mucosal surfaces as well as sterile body sites. Candida dubliniensis develops resistance to fluconazole (FLC) more rapidly than the closely related species C. albicans. The killing activity of amphotericin B (AMB), 5‐fluorocytosine (5FC), FLC, voriconazole (VRC) and posaconazole (POS) was determined against six C. dubliniensis clinical isolates, identified using molecular biological methods and C. dubliniensis CD36 reference strain. Minimum inhibitory concentrations (MICs) were determined using the Clinical and Laboratory Standards Institute standard procedure. Time‐kill assays were performed using RPMI‐1640 as test media over a 48‐h period. AMB proved to be fungicidal at ≥0.5 μg ml^?1 against all clinical isolates after 48 h. 5FC was only fungicidal at 32–64× MIC (4–8 μg ml^?1) against all C. dubliniensis isolates. FLC, VRC and POS were fungistatic; decrease in colony number was observed only at the highest concentrations tested (8, 4 and 4 μg ml^?1, respectively). Triazoles invariably showed fungistatic effect at concentrations attainable in the serum. In clinical situations when a fungicidal antifungal is desirable, AMB may be used.     

Adhesion of Candida parapsilosis to epithelial and acrylic surfaces correlates with cell surface hydrophobicity

We investigated in vitro adherence of 24 isolates of Candida parapsilosis and 12 isolates of Candida albicans with regard to their relative cell-surface hydrophobicity (CSH), adherence to human buccal epithelial cells (BEC) and acrylic surfaces. There was no significant interspecies difference in the relative adherence of C. parapsilosis and C. albicans isolates to BEC, although the former demonstrated a tendency for increased adherence. However, a significant intra-species variation in adherence among isolates of C. parapsilosis (P < 0.0001) to BEC, but not of C. albicans was noted. The superficial isolates of C. parapsilosis demonstrated a higher avidity (33%) to BEC than the systemic isolates. On regression analysis a significant positive correlation between C. parapsilosis adherence to BEC and denture acrylic surfaces was noted (r = 0.45, P = 0.02). Similarly, buccal cell adherence correlated strongly with CSH of C. parapsilosis (r = 0.63, P = 0.0008). These results shed further light on the intimate relationship between adherence and CSH in candidal colonization and imply that both C. parapsilosis and C. albicans are equally potent in colonizing mucosal surfaces with respect to the attributes investigated.     

The effect of brief exposure to sub‐therapeutic concentrations of chlorhexidine gluconate on germ tube formation of oral Candida dubliniensis

Adherence of Candida has been implicated as the first step in the pathogenesis of oral candidosis, and germ tube formation, a contributory attribute. While chlorhexidine gluconate is by far the most common antiseptic mouthwash prescribed in dentistry, its intraoral concentration fluctuates considerably because of the dynamics of the oral cavity. Hence, the main objective of this study was to investigate the effect of brief exposure to three different sub‐therapeutic concentrations of chlorhexidine gluconate on germ tube formation of Candida dubliniensis. Twelve oral isolates of C. dubliniensis were exposed to three different sub‐therapeutic concentrations of 0.005%, 0.0025% and 0.00125% chlorhexidine gluconate for 30 min. The antiseptic was removed, and following subsequent incubation in a germ tube inducing medium, the germ tube formation of these isolates was quantified microscopically. When compared with the controls, brief exposure to 0.005%, 0.0025% and 0.00125% chlorhexidine gluconate suppressed the ability to form germ tubes by 76.53% (P < 0.01), 49.17% (P < 0.01) and 3.45% (P > 0.05) respectively. These findings imply that brief exposure to sub‐therapeutic levels of chlorhexidine gluconate may modulate germ tube formation of C. dubliniensis, thereby suppressing its pathogenicity in vivo.     

Genotypic diversity and antifungal susceptibility of Cryptococcus neoformans isolates from paediatric patients in China

Cryptococcosis is a life‐threatening mycosis primarily occurring in adult patients particularly those with immunosuppression such as HIV infection/AIDS. The number of reported cases of paediatric cryptococcosis has increased in the last decade around the world, including China. However, current information on the characteristics of cryptococcosis in children, particularly the genotypic diversity and antifungal susceptibility of the isolates, is limited. In the present study, a total of 25 paediatric isolates of Cryptococcus neoformans were genotyped using the ISHAM‐MLST scheme. In vitro susceptibility to antifungal agents of the 22 isolates was tested using the CLSI M27‐A3 method. Our analyses revealed that the genotypic diversity of C. neoformans isolates from Chinese paediatric patients was low, with ST 5 (80%) and ST 31 (12%) being the two major sequence types. Reduced susceptibility to fluconazole (FLU), 5‐flucytosine (5‐FC) and itraconazole (ITR) was observed among C. neoformans isolates from Chinese paediatric patients, particularly among the ST5 isolates, which was similar to observations made on C. neoformans isolates from Chinese adult patients. In addition, the majority of isolates (3/4, 75%) obtained from deceased patients showed decreased antifungal susceptibility, which indicates that further monitoring of antifungal susceptibility of Cryptococcus isolates is warranted in management of paediatric cryptococcosis.     

Evaluation of virulence factors and antifungal susceptibility patterns of different Candida species isolated from the female camel (Camelus dromedarius) genital tract

The purposes of this study were to investigate the enzymatic activity of different Candida species and their antifungal susceptibility patterns. The study involved a total of 83 isolates of Candida from the genital tract of the female Camelus dromedarius. After species identification, the isolates were analysed for the production/activity of phospholipase, proteinase and haemolysin. In addition, the agar disc diffusion method was performed on the basis of CLSI guidelines M44‐A2 protocol for antifungal susceptibility testing. All the isolates were able to produce phospholipase, proteinase and haemolysin. A total of 35.48%, 87.09% and 64.51% of C. albicans isolates exhibited very high phospholipase, proteinase and haemolytic activities, respectively, whereas very high phospholipase, proteinase and haemolytic activities were determined in 5.76%, 23.07% and 45.16% of non‐C. albicans isolates respectively. Overall, 61 (73.5%) of Candida isolates were susceptible to fluconazole, 70 (84.3%) susceptible to clotrimazole, 82 (98.8%) susceptible to voriconazole, 76 (91.6%) susceptible to itraconazole, 75 (90.4%) susceptible to ketoconazole, 83 (100%) susceptible to amphotericin B, 81 (97.6%) susceptible to nystatin and 36 (43.4%) susceptible to flucytosine. Candida isolates showed higher haemolytic activity than that of other secreted hydrolases among vaginal Candida species. In addition, amphotericin B was the most in vitro effective antifungal drug and flucytosine had the poorest activity under such conditions.     

Evaluation of antifungal combination against Cryptococcus spp.

The second cause of death among systemic mycoses, cryptococcosis treatment represents a challenge since that 5‐flucytosine is not currently available in Brazil. Looking for alternatives, this study evaluated antifungal agents, alone and combined, correlating susceptibility to genotypes. Eighty Cryptococcus clinical isolates were genotyped by URA5 gene restriction fragment length polymorphism. Antifungal susceptibility was assessed following CLSI‐M27A3 for amphotericin (AMB), 5‐flucytosine (5FC), fluconazole (FCZ), voriconazole (VRZ), itraconazole (ITZ) and terbinafine (TRB). Drug interaction chequerboard assay evaluated: AMB + 5FC, AMB + FCZ, AMB + TRB and FCZ + TRB. Molecular typing divided isolates into 14 C. deuterogattii (VGII) and C. neoformans isolates were found to belong to genotype VNI (n = 62) and VNII (n = 4). C. neoformans VNII was significantly less susceptible than VNI (P = 0.0407) to AMB; C. deuterogattii was significantly less susceptible than VNI and VNII to VRZ (P < 0.0001). C. deuterogattii was less susceptible than C. neoformans VNI for FCZ (P = 0.0170), ITZ (P < 0.0001) and TRB (P = 0.0090). The combination FCZ + TRB showed 95.16% of synergistic effect against C. neoformans genotype VNI isolates and all combinations showed 100% of synergism against genotype VNII isolates, suggesting the relevance of cryptococcal genotyping as it is widely known that the various genotypes (now species) have significant impact in antifungal susceptibilities and clinical outcome. In difficult‐to‐treat cryptococcosis, terbinafine and different antifungal combinations might be alternatives to 5FC.     

Prevalence of Candida dubliniensis among cancer patients in Kuwait: a 5‐year retrospective study

Despite close genetic and phenotypic relationship of Candida dubliniensis with Candida albicans, its role in human disease is mostly restricted to oral colonisation, particularly among HIV‐infected patients. The prevalence of C. dubliniensis in association with other disease conditions has been infrequently reported. In this study, we present data on the prevalence of C. dubliniensis among yeast species isolated from cancer patients over a 5‐year period. A total of 1445 yeast isolates recovered from respiratory specimens, blood, urine and oral swabs were analysed. Candida dubliniensis isolates were provisionally identified by phenotypic methods and their identity was further confirmed by species‐specific amplification and/or sequencing of internally transcribed spacer region of rDNA. Antifungal susceptibility for fluconazole was determined by Etest. The number of isolates identified as C. dubliniensis, C. albicans and other yeast species were 71 (4.9%), 862 (59.6%) and 512 (35%) respectively. All the C. dubliniensis isolates originated from respiratory (5.9%) or oral (3.2%) specimens with an overall prevalence of 4.9%, and were found to be susceptible to fluconazole. The isolation of C. dubliniensis from respiratory or oral specimens and not from blood or urine specimens suggests that this species has preference to colonise these sites of human body.     

Experimental pathogenicity of Candida albicans and Candida dubliniensis with continuous and discontinuous fringes morphotypes

A possible correlation between the presence of discontinuous fringes and high virulence has been previously suggested. The aim of this study was to compare the pathogenicity of Candida albicans and Candida dubliniensis with continuous and discontinuous fringes morphotypes on mice. For C. albicans, two discontinuous fringe morphotype isolates (PN 69, PN 74), two continuous fringe morphotype isolates (N 60, N 33) and one reference strain were used. For C. dubliniensis, three discontinuous fringe morphotype isolates (97487, 97464, 97519), two continuous fringe morphotype isolates (97040, 98026) and one reference strain were used. Swiss male mice were inoculated with a standardised suspension of the microorganisms and observed for 35 days. The pathogenicity of the isolates was analysed according to parameters proposed previously. Three isolates were considered pathogenic: PN 74, N 60 and 98026. Strain N 60 killed the highest amount of mice (80%). Animals inoculated with C. albicans did not show differences on survival estimate. Candida dubliniensis 98026 was more pathogenic than samples 97464 and 97519. On the other hand, the sample 97487 showed a higher pathogenicity when compared with 97040 (Kaplan–Meier test, P = 0.008). Strains with continuous fringe morphotypes were also associated with Candida sp. virulence in vivo.     

Susceptibility of Malassezia pachydermatis to aminoglycosides

Previous studies have evaluated the action of gentamicin against Malassezia pachydermatis. The aim of this study was to evaluate in vitro susceptibility of M. pachydermatis to the aminoglycosides— gentamicin, tobramycin, netilmicin and framycetin. The minimum inhibitory concentration (MIC) of gentamicin was determined following methods M27‐A3 microdilution and Etest^®. The Etest^® was used to determine the minimum inhibitory concentration (MIC) of the tobramycin and netilmicin. The Kirby‐Bauer test was used to determine the antibiotic susceptibility to the framycetin. The MIC50 and MIC90 were 8.12 μg/mL and 32.5 μg/mL by microdilution method for gentamicin. The MIC50, determined by the Etest^®, was 8 μg/mL for gentamicin and netilmicin and 64 μg/mL for tobramycin. The MIC90 was 16 and 32 μg/mL for gentamicin and netilmicin respectively. The MIC90 was outside of the detectable limits for tobramycin. To framycetin, 28 strains (40%) of the 70 M. pachydermatis isolates tested showed a diameter of 22 mm, 22 strains (31.42%) showed a diameter of 20 mm, 16 strains showed a diameter of ≤ 18 mm, and only 5.71% of the isolates showed a diameter of ≥ 22 mm. This study provides evidence of high in vitro activity of the aminoglycosides—gentamicin, tobramycin, netilmicin and framycetin against M. pachydermatis. For gentamicin Etest^® showed similar values of MIC50 y MIC90 that the obtained by microdilution method. We considered Etest^® method could be a good method for these calculations with aminoglycosides.     

Species diversity,antifungal susceptibility and phenotypic and genotypic characterisation of Exophiala spp. infecting patients in different medical centres in Brazil

The Exophiala genus is responsible for many superficial and invasive infections resulting from black fungi. Identification of Exophiala at the species level is based on morphological observations complemented by molecular tests. The aim of this study was to identify 23 clinical isolates of Exophiala spp. and evaluate the antifungal susceptibility to seven different agents. Molecular identification was based on an analysis of ITS region of rDNA using genomic databases. The micromorphology was evaluated by microculture and scanning electron microscopy. The susceptibility tests were performed using the antifungal agents 5‐fluorocytosine (5‐FC), amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), posaconazole (PSC), caspofungin (CFG) and terbinafine (TRB). The ITS analysis identified 100% of the following isolates as: E. dermatitidis (8), E. xenobiotica (6), E. bergeri (4), E. oligosperma (3), E. spinifera (1) and E. mesophila (1). The antifungal susceptibility tests showed that the triazoles compounds were in vitro the most active agents against Exophiala. ITS sequencing enabled the accurate identification of the 23 tested isolates. The triazoles, particularly itraconazole and posaconazole, exhibited MIC values lower than AMB, CAS and 5‐FC. Although the guidelines do not indicate AMB for treatment against Exophiala spp., this study showed activity for all of the tested species, except E. mesophila.     

Isolation of Rhodotorula mucilaginosa from blood cultures in a tertiary care hospital

Rhodotorula species have traditionally been considered as one of common non‐virulent environmental inhabitant. They have emerged as an opportunistic pathogen, particularly in immunocompromised hosts and most infections have been associated with intravenous catheters in these patients. We review the isolates in blood cultures of Rhodotorula mucilaginosa in our Hospital. We describe the demographic and clinical features of the cases and the antifungal susceptibility profiles of the isolates. Selected patients had an isolation of R. mucilaginosa in blood cultures in our tertiary care Hospital. All data were collected retrospectively from clinical records during 5 years. We report 8 isolates in blood, two of them were considered contaminants. Immunosuppression, surgery, previous antibiotic therapy were common clinical features. For all the isolates, minimum inhibitory concentration (MIC) values were high for echinocandins and azoles and low for amphotericin B and 5‐flucytosine. One strain showed atypical susceptibility profile. Rhodotorula mucilaginosa may be present on the skin and blood cultures can be contaminated. Fungaemia due to R. mucilaginosa is a rare clinical entity which requires risk factors but clinically relevant because of the multiresistant profile. Rhodotorula mucilaginosa shows high MIC values for azoles and echinocandins, therefore amphotericin B and flucytosine must be administered as antifungal therapy.     

Comparison of fks gene mutations and minimum inhibitory concentrations for the detection of Candida glabrata resistance to micafungin: A systematic review and meta‐analysis

Candida resistance to antifungals impaired invasive candidiasis outcome. In a context of echinocandin resistance development, we aimed to evaluate the association between phenotypic resistance to micafungin and fks mutations of Candida glabrata. For this systematic review and meta‐analysis, we searched MEDLINE, Scopus and Web of Science for reports published up to December 2017. Studies of C glabrata candidiasis with minimum inhibitory concentrations (MIC) determination of micafungin and fks genotyping were included. Reviews, studies not using reference methods, non‐glabrata Candida, experimental isolates and undetailed mutations were excluded. Two authors independently assessed the eligibility of articles and extracted data. The main outcome was the diagnostic accuracy of fks mutations compared to micafungin MIC for C glabrata, measured as fixed‐effect odd ratio. Heterogeneity was calculated with the I² statistic. This study is registered with PROSPERO (CRD42018082023). Twenty‐four studies were included in the meta‐analysis. Pooled analysis found that S663P (OR 7.25, 95% CI 3.50‐15.00; P < 0.00001), S629P (OR 3.70, 1.64‐8.33; P = 0.002) and F659del (OR 5.66, 1.22‐26.18; P = 0.03) were associated with increased risk of having a resistant isolate according to authors' interpretation of MICs. In sensitivity analysis based on new CLSI clinical breakpoints, the ORs for S663P and S629P remained significant. Genotyping of isolates of C glabrata for S663P and S629P mutations is an effective alternative to micafungin susceptibility tests. Relevant molecular markers of drug resistance will significantly improve the management of C glabrata infections.     

The role of drug efflux pumps in Malassezia pachydermatis and Malassezia furfur defence against azoles

This study aims to evaluate the effect of efflux pump modulators (EPMs) on the minimal inhibitory concentration (MIC) of fluconazole (FLZ) and voriconazole (VOR) in Malassezia furfur and Malassezia pachydermatis. The in vitro efficacy of azoles, in combination with EPMs (ie haloperidol‐HAL, promethazine‐PTZ and cyclosporine A‐CYS), against 21 M. furfur from bloodstream infection patients and 14 M. pachydermatis from the skin of dogs with dermatitis, was assessed using a broth microdilution chequerboard analysis. Data were analysed using the model‐fractional inhibitory concentration index (FICI) method. The MIC of FLZ and VOR of Malassezia spp. decreased in the presence of sub‐inhibitory concentrations of HAL and/or PTZ. The synergic effect was observed only in strains with FLZ MIC≥128 μg/mL for M. furfur, FLZ MIC≥64 μg/mL for M. pachydermatis and VOR MIC≥4 μg/mL in both Malassezia spp. These results suggest that the drug efflux pumps are involved as defence mechanisms to azole drugs in Malassezia yeast. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Finally, the above FLZ and VOR MIC values might be considered the cut‐off to discriminate susceptible and resistant strains.     

Phospholipase and proteinase activities of Candida isolates from denture wearers

The aim of the present study was to characterise phospholipase and proteinase activities of oral Candida isolates from 100 denture wearers and to study the relationship of these activities with denture stomatitis. Of 100 patients studied, 44 suffered from denture stomatitis. Specimens were collected by swabbing the denture and underlying mucosa. Isolates were previously identified by conventional mycological and genotypic methods. The phospholipase and proteinase activities were evaluated by agar plate methods. A total of 152 isolates were recovered from denture and underlying mucosa, including 101 Candida albicans, 18 Candida tropicalis, 14 Candida glabrata, 11 Candida guilliermondii, four Candida parapsilosis, two Saccharomyces cerevisiae and one isolate each of Candida dubliniensis and Candida krusei. Most C. albicans (97%) showed phospholipase activity; furthermore, the unique C. dubliniensis isolate showed a moderate phospholipase activity. The isolation of C. albicans (chi‐square test, P = 0.0016) and phospholipase production by Candida spp. (chi‐square test, P = 0.0213) was found to be significantly associated with denture stomatitis. Proteinase production was observed in <30% of isolates, and it was not related to the presence of denture stomatitis (P = 0.7675). Candida albicans isolates may produce both virulence factors, although the proteinase production was only observed in <30% of the isolates. Phospholipase production was exclusive of C. albicans and C. dubliniensis.     

Investigation of minor species Candida africana,Candida stellatoidea and Candida dubliniensis in the Candida albicans complex among Yaoundé (Cameroon) HIV‐infected patients

Minor species of the Candida albicans complex may cause overestimation of the epidemiology of C. albicans, and misidentifications could mask their implication in human pathology. Authors determined the occurrence of minor species of the C. albicans complex (C. africana, C. dubliniensis and C. stellatoidea) among Yaoundé HIV‐infected patients, Cameroon. Stool, vaginal discharge, urine and oropharyngeal samples were analysed by mycological diagnosis. Isolates were identified by conventional methods and mass spectrometry (MS; carried out by the matrix‐assisted laser desorption–ionisation time‐of‐flight MS protocol). Candida albicans isolates were thereafter submitted to the PCR amplification of the Hwp1 gene. The susceptibility of isolates to antifungal drugs was tested using the Clinical and Laboratory Standards Institute M27‐A3 protocol. From 115 C. albicans obtained isolates, neither C. dubliniensis nor C. stellatoidea was observed; two strains of C. africana (422PV and 448PV) were identified by PCR electrophoretic profiles at 700 bp. These two C. africana strains were vaginal isolates. The isolate 448PV was resistant to ketoconazole at the minimal inhibitory concentration of 2 μg ml^?1, and showed reduced susceptibility to amphotericin B at 1 μg ml^?1. This first report on C. africana occurrence in Cameroon brings clues for the understanding of the global epidemiology of this yeast as well as that of minor species of the C. albicans complex.     

Prevalence of biofilm formation in clinical isolates of Candida species causing bloodstream infection

Candida species are the fourth most common cause of nosocomial invasive infections. Biofilm formation is recognised as one virulence factor of Candida species. A total of 243 Candida albicans, 81 C. glabrata, 33 C. parapsilosis, 14 C. dubliniensis, 8 C. tropicalis, 8 C. lusitaniae, 5 C. krusei and 1 C. pelliculosa isolates causing bloodstream infections were evaluated for biofilm formation. The biofilm formed on silicone elastomer preincubated with human serum was quantified by estimation of the metabolic activity through XTT assay and visualised by light and scanning electron microscopy. Forty per cent of the C. albicans isolates formed biofilm compared to 88.7% of the non‐albicans Candida isolates (P < 0.0001). Among non‐albicans Candida spp., biofilm formation was most commonly observed in C. tropicalis and C. lusitaniae (100%), followed by C. glabrata (95%), C. dubliniensis (85.7%) and C. parapsilosis (66.7%). A quantitative correlation was observed between the amount of biofilm observed microscopically, and that determined by metabolic activity measurements. The biofilms of all Candida species were composed of basal yeast cells with the exception of C. parapsilosis which produced biofilms consisting of pseudohyphae and aggregated yeast cells. These results suggest that biofilm formation as a virulence factor might have a higher significance for non‐albicans Candida species than for C. albicans.