Antifungal susceptibility and genotypes of Candida albicans strains from patients with vulvovaginal candidiasis

Studies of the genetic diversity of Candida albicans strains and the correlation between the antifungal susceptibility and gene diversity of C. albicans were carried out and the results were found to be inconsistent. To investigate antifungal susceptibility and genotypes of C. albicans strains from patients with vulvovaginal candidiasis (VVC), the genotypes of C. albicans in patients with VVC were studied using a recently developed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) of CAI microsatellite method and antifungal susceptibility was tested using E-test methods. Twenty-six genotypes were identified from 89 strains of C. albicans isolated from patients with VVC. Candida albicans isolates were susceptible to amphotericin B, flucytosine, ketoconazole and fluconazole. The dominant genotypes (A, B, C, D) account for 69.7% (62/89) of C. albicans . The resistant rate of C. albicans genotype B to itraconazole (ITR) and that of C. albicans non-genotype B strains were 66.7% (14/21) and 4.4% (3/68) respectively at P  < 0.05. We concluded that C. albicans genotype B from patients with VVC was more resistant to ITR.     

Resveratrol and its antifungal activity against Candida species

Resveratrol is a natural stilbene synthesised by plants. This compound has been shown to inhibit the growth of Candida albicans TIMM 1768 efficiently. Till date, no information is available for other Candida species. The evaluation of the antimicrobial activity of resveratrol was analysed by the inhibition of the growth and metabolism assays. Our data indicate that resveratrol is not effective against Candida albicans and non‐C. albicans species (C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei) in vitro. The potential candidacidal activity could not be confirmed.     

Repeated exposure of Candida spp. to miconazole demonstrates no development of resistance

Oropharyngeal candidiasis (OPC) is a common infection among the immuno‐compromised population. Treatments include both systemic azoles, most commonly fluconazole (FLU), and topical agents such as miconazole (MICON). However, resistance to FLU has been reported with a greater frequency. The aim of this study was to determine the potential for development of resistance following repeated exposure of Candida spp. to MICON. Two clinical isolates each of Candida albicans, C. glabrata, and C. tropicalis were tested. Fifteen passages of each strain were performed in concentrations of MICON at 0.5 minimum inhibitory concentration (MIC), 1 MIC, 2 MIC and 4 MIC, with MIC determinations performed on growth obtained following each passage. There was no increase in the MIC of four of the six strains following fifteen passages in MICON. One C. albicans strain demonstrated a four‐five dilution increase in MICON MIC at all concentrations and one C. glabrata strain showed a fivefold MICON MIC increase when exposed to 4 MIC. Although an increase in MIC was noted in these two isolates, the MICON MIC was still very low (0.5 μg ml^?1). In general, there was no increase in MIC demonstrated by repeated exposure to MICON in this study.     

肿瘤患者合并肺部念珠菌感染药敏测定研究

采用ＲＰＭＩ１６４０及ＦＤＡ抗生素３号两种培养基的微量稀释法对肿瘤患者合并肺感染分离出５１株念珠菌进行药敏对比测定。结果显示二性霉素Ｂ在两种培养基中均具有非常好的抗菌活性，敏感率均为９８．０％，其次酮康唑也显示出良好的抗菌活性，敏感率均为９４．１％。相比之下，在ＲＰＭＩ培养基中伊曲康唑和氟康唑的敏感性稍差，敏感率分别为９２．２％和９０．２％，但在ＦＤＡ抗生素３号培养基中，伊曲康唑和氟康唑敏感性明显下降，敏感率分别为７０．６％和６６．７％。为此我们建议抗真菌药敏试验，尤其是咪唑类药物应该选用ＮＣＣＬＳ推荐的ＲＰＭＩ１６４０培养基做药敏试验     

Antifungal susceptibility of clinical Candida parapsilosis isolates in Kuwait

This study presents data on antifungal susceptibility of 114 Candida parapsilosis isolates recovered from clinical specimens in Kuwait. Candida parapsilosis isolates originating from blood ( n  = 66) and other clinical specimens ( n  = 48) were tested by Etest against amphotericin B (AP), caspofungin (CS), 5-flucytosine (FC), fluconazole (FL) and voriconazole (VO). The plates were incubated at 35 °C and readings for minimum inhibitory concentrations (MIC) were recorded after 24 and 48 h of incubation. The MIC ranges and MIC₉₀ read after 48 h were as follows: 0.064–1 and 0.5 μg ml⁻¹ for AP; 0.125–4 and 1.5 μg ml⁻¹ for CS; 0.047 to >256 and 1 μg ml⁻¹ for FL; 0.023 to >32 and 0.125 μg ml⁻¹ for FC and <0.002–1 and 0.047 μg ml⁻¹ for VO respectively. According to Clinical Laboratory Standards Institute criteria, all the isolates were susceptible to VO, and resistance against FC and FL was <2%. Eight (7%) isolates exhibited reduced susceptibility (MIC >1 μg ml⁻¹) to CS including six isolates with MIC of ≥2 μg ml⁻¹ at 48 h reading. The antifungal resistance among bloodstream isolates of C. parapsilosis against AP, FL, FC and VO in Kuwait is rare. This is the first report on CS susceptibility of C. parapsilosis isolates from Arabian Gulf region.     

Antifungal susceptibility testing of Candida albicans by flow cytometry

Antifungal susceptibilities of 28 Candida albicans isolates and two quality control strains to amphotericin B and fluconazole were determined by flow cytometry and microdilution method. Minimum inhibitory concentrations (MICs) obtained by flow cytometry were compared with the results obtained by The National Committee for Clinical Laboratory Standards Subcommittee (NCCLS) broth microdilution method. The agreement of results (within two dilution) obtained was found as 96 and 93% for amphotericin B and fluconazole, respectively. At least 24 h incubation was required for reading the microdilution assays. Four hours of incubation was required for fluconazole, whereas 2-h incubation was sufficient for amphotericin B to provide MIC by flow cytometry. Results of this study show that flow cytometry provides a rapid and sensitive in vitro method for antifungal susceptibility testing of Candida albicans isolates.     

Antifungal activity of silver nanoparticles in combination with nystatin and chlorhexidine digluconate against Candida albicans and Candida glabrata biofilms

Although silver nanoparticles (SN) have been investigated as an alternative to conventional antifungal drugs in the control of Candida‐associated denture stomatitis, the antifungal activity of SN in combination with antifungal drugs against Candida biofilms remains unknown. Therefore, the aim of this study was to evaluate the antifungal efficacy of SN in combination with nystatin (NYT) or chlorhexidine digluconate (CHG) against Candida albicans and Candida glabrata biofilms. The drugs alone or combined with SN were applied on mature Candida biofilms (48 h), and after 24 h of treatment their antibiofilm activities were assessed by total biomass quantification (by crystal violet staining) and colony forming units enumeration. The structure of Candida biofilms was analysed by scanning electron microscopy (SEM) images. The data indicated that SN combined with either NYT or CHG demonstrated synergistic antibiofilm activity, and this activity was dependent on the species and on the drug concentrations used. SEM images showed that some drug combinations were able to disrupt Candida biofilms. The results of this study suggest that the combination of SN with NYT or CHG may have clinical implications in the treatment of denture stomatitis. However, further studies are needed before recommending the use of these drugs safely in clinical situations.     

Antifungal activity of propolis on different species of Candida

Propolis is a resinous material collected by bees from the buds or other parts of plants. It is known for its biological properties, having antibacterial, antifungal and healing properties. The antifungal activity of propolis was studied in sensitivity tests on 80 strains of Candida yeasts: 20 strains of Candida albicans, 20 strains of Candida tropicalis, 20 strains of Candida krusei and 15 strains of Candida guilliermondii. The yeasts showed a clear antifungal activity with the following order of sensitivity: C. albicans > C. tropicalis > C. krusei > C. guilliermondii. Patients with full dentures who used a hydroalcoholic propolis extract showed a decrease in the number of Candida.     

Resistance of Candida albicans to direct lethal miconazole action induced by low-level miconazole

Logarithmic phase cells of Candida albicans are susceptible to physicochemical damage by greater than or equal to 2 x 10(-5)mol miconazole resulting in direct lethal action (DLA). Stationary phase yeasts are resistant to such action. At low levels (i.e. less than 10(-5)mol), miconazole can inhibit synthesis of cell membrane components and can also revert DLA-susceptible cells to DLA resistance. Reversion was studied in relation to low-level miconazole exposure time. DLA was assessed by viable count determinations. Most cells in susceptible cultures reverted to resistance within 30 to 60 min of exposure to 2.0 x 10(-6)mol miconazole. The rapidity of this shift suggested subtle alterations in existing cell membrane material in response to low-level miconazole. Saturation of membrane fatty acids might explain the shift.     

Intrinsic in vitro susceptibility of primary clinical isolates of Aspergillus fumigatus, Aspergillus terreus, Aspergillus nidulans, Candida albicans and Candida lusitaniae against amphotericin B

A total of 60 clinical fungal isolates from patients without prior amphotericin B treatment and three control strains were evaluated for their intrinsic susceptibility to amphotericin B (AmB) using microdilution, Etest and disc diffusion assays, on three media each, Roswell Park Memorial Institute (RPMI) 1640, Antibiotic Medium 3 (AM3) and High Resolution Medium. The fungal strains included isolates of Aspergillus fumigatus (n = 10), Aspergillus terreus (n = 12), Aspergillus nidulans (n = 9), Candida albicans (n = 6) and Candida lusitaniae (n = 23). The A. terreus strains were significantly less susceptible to AmB than the A. fumigatus strains in all nine experimental settings (P-values ranging from 0.009 to <0.00001). The A. nidulans strains were equally susceptible to AmB as the A. fumigatus strains in seven of nine experimental settings and less susceptible in two (microdilution performed on RPMI and AM3, P = 0.01 and 0.007). The C. lusitaniae strains were equally susceptible to AmB as the C. albicans strains in seven of nine experimental settings and more susceptible in two (microdilution and Etest, both performed on AM3, P = 0.01 and 0.0002). Thus, we confirmed that A. terreus is intrinsically less susceptible to AmB than A. fumigatus. In contrast, nine German clinical isolates of Aspergillus nidulans were found equally susceptible to AmB as 10 isolates of A. fumigatus. Furthermore, we found 23 German clinical isolates of C. lusitaniae from patients without prior treatment with AmB equally susceptible to AmB as C. albicans.     

Antifungal activity of tri‐ and tetra‐thioureido amino derivatives against different Candida species

The in vitro antifungal activity of six thioureido substituted amines (P1–P6) was evaluated against Candida species, including Candida albicans, C. glabrata, C. krusei and C. parapsilosis. These tri‐ and tetra‐thioureido amino derivatives with different methylation levels were synthesised through easy synthetic routes to evaluate their antifungal properties against Candida species. Among all studied derivatives, the tri‐(2‐thioureido‐ethyl)‐amine (P1) was the most active compound inhibiting C. albicans and C. glabrata at a concentration of 0.49 μg ml^?1; P3, the N,N′,N′′,N′′′‐hexamethyl‐derivative, also showed inhibitory activity against C. albicans and C. glabrata, but in higher concentrations (250 μg ml^?1). The N,N′,N′′,N′′′‐tetramethylated amine (P5) only inhibited the growth of C. glabrata, but its corresponding N,N′,N′′,N′′′‐octamethyl derivative (P6) was also active against C. glabrata (125 μg ml^?1) and it was the only compound active against C. parapsilosis. P2 and P4 showed no significant antifungal activity. The structure–activity relationship of the thioureido‐substituted derivatives indicates that the molecular branching and the alkylation levels can influence the antifungal activity. This study demonstrated that thioureido derivatives exhibited significant antifungal activity against Candida species and that they can be considered as a very promising bioactive lead compound to develop novel antifungal agents.     

Microbial profile and drug resistance of Candida strains isolated from the blood of children: an 11-year study

The latest observations indicate a continuous increase in the frequency of fungal infections, particularly in hospital patients, accompanied by changes in both the profile of the isolated strains and their drug susceptibility. The objective of this study was to evaluate the trend in the incidence of candidaemia and susceptibility of antifungal drugs in the Polish Mother's Memorial Hospital over an 11-year period. Blood samples taken from the hospitalised children were sent to the Department of Clinical Microbiology for diagnostic purposes. A total of 195 Candida strains were isolated: 47.7% Candida albicans and 52.3% non- albicans . Candida parapsilosis was isolated in 65.7% of non- albicans strains. The prevalence of Candida spp. decreased from 16.9–20.5% in the years 1996–1997 to 3.1–2.1% in the years 2005–2006. In the years 2000–2005, non- albicans strains were more prevalent. All C. albicans strains were susceptible to amphotericin B, 2.94% of non -C. albicans strains were semisusceptible to amphotericin B, 98.92% of C. albicans and 85.29% of non- albicans strains were susceptible to 5-fluorocytosine. Candida spp. strains are predominant pathogens in fungaemia in children in our hospital. Over the last few years, C. albicans have been replaced by non- albicans strains (predominantly C. parapsilosis ), which exhibit a higher level of drug resistance. The number of Candida spp. isolated from blood decreased during the 11-year study.     

Influence of serum on in vitro susceptibility testing of echinocandins for Candida parapsilosis and Candida guilliermondii

Echinocandins are antifungal drugs used for the treatment of invasive candidiasis and aspergillosis. They bind to serum proteins within a rate of 96 to >99%. The effect of serum on in vitro echinocandin susceptibility tests of certain Candida and Aspergillus species was reported. This study was performed to determine the effect of human serum on in vitro susceptibility testing of echinocandins for clinical isolates of Candida parapsilosis and Candida guilliermondii, the species which generally have higher minimum inhibitor concentrations compared with other Candida species. One hundred C. parapsilosis and 20 C. guilliermondii isolates were included in the study. The susceptibility tests of caspofungin, micafungin and anidulafungin were performed using microdilution method, either in the presence or absence of 50% human serum, according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines. It was demonstrated that human serum significantly affects the in vitro susceptibility results of echinocandins for C. parapsilosis and C. guilliermondii isolates, mostly yielding an increase in MICs. The most prominent fold changes were for micafungin and anidulafungin in C. parapsilosis, and for anidulafungin in C. guilliermondii isolates. Serum influences the in vitro echinocandin susceptibility in C. parapsilosis and C. guilliermondii. The mechanism and clinical significance of this in vitro change need to be clarified.     

Posaconazole susceptibility testing against Candida species: comparison of broth microdilution and E-test methods

Posaconazole (POS) is a newer triazole with activity against yeasts and moulds. POS and fluconazole were tested in vitro against 32 Candida albicans, 30 C. glabrata, 21 C. tropicalis, 29 C. krusei, 28 C. parapsilosis, 50 C. inconspicua, 13 C. kefyr and 5 C. famata isolates using CLSI broth microdilution method (BMD). We compared E-test and a modified BMD using polyethylene-glycol (PEG) as solvent to the CLSI method. BMDs and E-test were performed according to CLSI and the manufacturer's instructions respectively. Geometric means of POS MICs using BMD were 0.71, 0.22 and 0.21 microg ml(-1) against C. glabrata, C. krusei and C. inconspicua, respectively, and remained below 0.1 microg ml(-1) against all other species tested. One of two C. albicans and two of three C. glabrata isolates resistant to fluconazole showed MICs above 8 microg ml(-1) to POS. The impact of using PEG instead of DMSO had only a minor effect (agreements above 95% with the exception of C. parapsilosis). E-tests read after 24 h showed good agreement with the BMD. POS exhibited excellent in vitro activity against Hungarian Candida strains. E-test showed good correlation with the CLSI method, but to facilitate the comparability of results we believe that DMSO should be used as solvent in the BMD.     

Potent anti-microbial activity of traditional Chinese medicine herbs against Candida species

Anti-candidial activities of eight traditional Chinese medicinal (TCM) herbs were evaluated against six different Candida species. TCM preparations were screened for antifungal activity using a standard agar diffusion assay. Following identification of potential candidate herbs, their minimum inhibitory concentration (MIC) values were determined using the standardised NCCLS M-27A broth microdilution assay. Among TCM herbs, Rhizoma Coptidis had potent antifungal activity against Candida glabrata, Candida krusei and Candida tropicalis, but not against Candida albicans, Candida dubliniensis and Candida parapsolosis. The MIC values of the Rhizoma Coptidis against C. glabrata, C. krusei and C. tropicalis were 50, 50 and 100 microg ml(-1) respectively. We report here, for the first time, the potent antifungal activity of Rhizoma Coptidis and Cortex phellodendri Chinesis on three different non-albicans Candida species, C. glabrata, C. krusei and C. tropicalis and hence their possible use as therapeutic agents.     

In vitro activity of nitroxoline against clinical isolates of Candida species

The in vitro antifungal activity of the quinoline nitroxoline has been compared with those of amphotericin B, flucytosine, and two azoles, miconazole and ketoconazole, against clinical isolates of Candida spp. A total of 186 isolates of 10 species of Candida and two culture collection strains were tested by an agar-dilution technique. Nitroxoline was highly active against Candida spp. MICs for nitroxoline ranged between 0.25-2 micrograms ml-1 for 186 representative strains. With MIC90 as the measure of antifungal activity, nitroxoline appeared to be superior to the imidazoles studied. Data for individual species of Candida revealed that the activities of nitroxoline and amphotericin B were generally just as effective against C. albicans, whereas flucytosine was the most active agent against Candida spp.     

Standardized molecular typing of Candida albicans strains

Summary. A method is presented for the standardization of Candida albicans DNA fingerprinting, which is based on Southern hybridization of Eco RI-digested chromosomal DNA with the moderately repetitive DNA element CARE-2 and the subsequent rehybridization of the blots with a molecular size marker also included in each DNA sample. This method resulted in extremely precise alignment of all strain-specific CARE-2 hybridization patterns, even when analysed on different gels, and will enhance the accuracy of genetic relationship determinations in epidemiological studies including large numbers of strains.
Zusammenfassung. Zur Standardisierung des DNA-Fingerprinting von Candida albicans wurde eine Methode entwickelt, die auf der Southern Hybridisierung Eco RI-gespaltener chromosomaler DNA mit dem mittelrepetitiven DNA-Element CARE-2 und der darauffolgenden Rehybridisierung der Blots mit einem auch in den Proben enthaltenen molekularen Größenmarker beruht. Dies resultierte in einer äußerst präzisen Größen-bestimmung der hybridisierenden Fragmente, so daß alle stammspezifischen CARE-2-Hybridisierungsmuster exakt verglichen werden konnten, auch wenn die Isolate auf verschiedenen Gelen analysiert wurden. Die Methode erhöht die Genauigkeit der Bestimmung genetischer Verwandtschaftsbeziehungen in epidemiologischen Untersuchungen, in denen eine große Anzahl von Stämmen analysiert wird.     

Susceptibility to antifungal agents of Candida species isolated from paediatric and adult patients with haematological diseases

Summary The susceptibility to six antifungals: amphotericin B (AMF), 5-fluorocytosine (5-F), miconazole (MIK), ketoconazole (KET), fluconazole (FLU) and itraconazole (ITR) was tested among 206 Candida spp. isolated from paediatric and adult patients with haematological malignancies. To determinate the susceptibility the commercial microdilution method Fungitest (Bio-Rad, France) was used. The strains were classified as susceptible, intermediate susceptible, or resistant on the base of the growth in following breakpoint concentrations of particular drugs: 2 and 8 microg ml(-1) for AMF, 2 and 32 microg ml(-1) for 5-F, 0.5 and 8 microg ml(-1) for MIK, 0.5 and 4 microg ml(-1) for KET and ITR, and 8 and 64 microg ml(-1) for FLU. The highest activity to overall species showed AMF (only one resistant strain) and 5-F (85% susceptible strains). Most of C. albicans isolates were susceptible to tested azoles. The percentages of C. albicans resistant to FLU, ITR, KET and MIK were 4, 11, 8, and 0.8%, respectively. The less susceptible to azoles were C. glabrata and C. krusei (14% and 44% isolates resistant to FLU). A non-albicans Candida isolated from adult patients receiving KET prophylaxis was more frequently resistant to FLU than isolates from patients without previous exposure to azoles (P < 0.05). We did not observe differences in the susceptibility of Candida strains isolated from children compared with those from adults.     

Efficacy of anidulafungin against biofilms of different Candida species in long-term trials of continuous flow cultivation

Long-term continuous flow culture allows the investigation of dynamic biofilms under microaerophilic or aerobic conditions. We studied the biofilm formation and changes of susceptibility in 30 blood culture isolates (48 experiments) of different Candida species exposed to anidulafungin in 0.16 ml or 7.7 ml flow chambers. The flow rate (F) was adjusted to a very low rate of 1.3 ml h(-1) resulting in an exchange rate of up to 180 and 6.25 times chamber volumes per 24 hours in the small and large chambers, respectively. The results of culture at a very low flow rate were markedly different from cultures in micro well plates. Low flow rates may better mimic the in vivo situation and thus may be of higher relevance for the clinical setting. Under these conditions, a general resistance of fungal biofilms against anidulafungin cannot be confirmed. Strains of C. albicans and C. glabrata showed very uniform results whereas the C. parapsilosis group and C. lusitaniae varied from high susceptibility to resistance. Species differentiation of the C. parapsilosis group appears to be appropriate in clinical microbiological diagnostics. For the majority of the tested Candida species, anidualafungin was more effective than voriconazole. For the species C. lusitaniae and C. guilliermondii susceptibility testing should be considered prior to clinical use of echinocandin antifungals.     

Inhibitory activity of thymol against the formation and viability of Candida albicans hyphae

As the capacity of Candida albicans to produce hyphae is considered an important virulence factor in the pathogenesis of candiasis, the aim of this study was to investigate whether thymol, the major component of thyme oil, can interfere with the filamentous forms of Candida albicans and their viability. The morphological transition from yeasts to filamentous forms was investigated by analysing the morphological index (MI), which classifies the differentiated forms and blastoconidia; viability was investigated by means of fluorescence microscopy using a new SYTO-9 and propidium iodide method previously used to stain only blastoconidia. Without thymol, there was an average of 94.00 +/- 3.06% hyphal forms. After 6 h of incubation with 1x MIC (125 microg ml(-1)), 1/2x MIC and 1/4x MIC of thymol, filamentation was, respectively, 14.33 +/- 8.25%, 28.33 +/- 7.17% and 45.67 +/- 8.09% in comparison with control (all statistically significant). In the absence of thymol, viable cells accounted for an average of 93.00 +/- 4.00% whereas, after 6 h of incubation with 1x MIC, 1/2x MIC and 1/4x MIC of thymol, the presence of 54.33 +/- 1.86%, 29.00 +/- 3.61% and 23.00 +/- 2.52% of yellow-orange coloured forms indicated damaged membranes and reduced viability. Our findings show that thymol interferes with the formation and viability of hyphae. This can be attributed to the characteristics of thymol disturbing Candida cell membranes and metabolism, probably by affecting fungal cell-wall synthesising enzymes.