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.     

Molecular epidemiology of Candida species isolated from urine at an intensive care unit

Candida spp. has been the leading microorganism isolated from the urine specimens of patients hospitalized at the Anesthesiology and Reanimation intensive care unit (ICU) of Dokuz Eylul University Hospital, Izmir, since 1998. This study was undertaken to investigate the clonal relationship of Candida urine isolates in order to find the mode of spread among the patients. Epidemiological surveillance of 38 Candida albicans, 15 Candida tropicalis and 12 Candida glabrata recovered from the urine specimens of patients who were hospitalized in the ICU between June 11, 2000 and October 15, 2001 was carried out by antifungal susceptibility testing and randomly amplified polymorphic DNA (RAPD) analysis. Two short primers [Cnd3 (5'-CCAGATGCAC-3') and Cnd4 (5'-ACGGTACACT-3')] were used for RAPD. None of the isolates had high minimal inhibitory concentration (MIC) values (>1 microg ml(-1)) against amphotericin B with MIC50 values of 0.5 microg ml(-1), 0.5 microg ml(-1) and 0.125 microg ml(-1) for C. albicans, C. tropicalis and C. glabrata isolates, respectively. However, three C. glabrata isolates were resistant and one C. albicans and five C. glabrata isolates were dose-dependent susceptible (D-DS) to fluconazole. Among C. albicans isolates 19 and 20 patterns were detected with primers Cnd3 and Cnd4, respectively. When primers Cnd3 and Cnd4 were evaluated together, three and four genotypes were identified for C. tropicalis and C. glabrata isolates, respectively. Our results suggest that the source of C. albicans isolates was mostly endogenous. It is difficult to interpret the mode of spread of C. tropicalis and C. glabrata urine isolates as we obtained insufficient banding patterns for these species.     

Antifungal activity of miconazole against recent Candida strains

Miconazole (MICON) has long been used for the topical treatment of mucosal candidiasis. However, the preponderance of MICON susceptibility data was generated before standard methodology was established, and prior to the emergence of fluconazole (FLU)‐resistant strains. The objective of this study was to determine the antifungal activity of MICON and comparators against recent clinical isolates of Candida spp. using standard Clinical and Laboratory Standards Institute methodology. One hundred and fifty isolates, consisting of 25 strains each of Candida albicans, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis and C. dubliniensis, were tested. Of these, twenty‐two strains were known to be FLU‐resistant. Minimum inhibitory concentrations (MICs) were determined for MICON, amphotericin B (AM), caspofungin (CAS), clotrimazole (CLOT), FLU, itraconazole (ITRA), nystatin (NYS) and voriconazole (VOR). MICON demonstrated potent inhibitory activity against all of the strains tested. The MIC₉₀ for MICON was 0.12 μg ml^?1 against FLU‐susceptible strains, which was comparable to that of AM, CAS, CLOT, ITRA and VOR. The MICON MIC₉₀ against FLU‐resistant strains was 0.5 μg ml^?1, which was 12‐fold lower than the FLU MIC₉₀. Our study showed that MICON possesses potent activity against all of the Candida isolates tested, including those with known FLU resistance. This indicates that recent clinical isolates remain susceptible to this antifungal and that MICON could be used as first‐line treatment for oropharyngeal candidiasis.     

Antifungal drug susceptibility of Cryptococcus neoformans from clinical sources in Nairobi, Kenya

The serotypes and mating types of 80 clinical isolates of Cryptococcus neoformans from Kenya were studied and subjected to broth microdilution susceptibility testing to amphotericin B (AMP), flucytosin, fluconazole (FLC), itraconazole (ITC) and miconazole (MCZ). The isolates included C. neoformans var. grubii- 75 of 80 (serotype A; 93.7%), C. neoformans var. neoformans- three of 80 (3.8%) and C. neoformans var. gattii- two (serotype B; 2.5%). Mating experiment confirmed all the isolates to be alpha-mating type. Seventy-eight (97.5%) of the isolates had minimum inhibitory concentration (MIC) of < or =0.5 microg ml(-1) to AMP and at 1 microg ml(-1), 100% of the isolates were inhibited. Flucytosin resistance was observed in 21% with MIC in which 90% of the isolates were inhibited (MIC90) of 64 microg ml(-1). Only 23.8% of the strains were susceptible to FLC with 65% susceptible dose-dependent (SDD) and 11.2% resistant. Itraconazole susceptibility was 61.3% while the rest were either SDD or resistant. The MIC90 for ITC and MCZ were 0.5 and 2 microg ml(-1) respectively. The study reports the serotypes, mating types and highlights the existence of azoles resistance in C. neoformans in Nairobi which calls for antifungal drug resistance surveillance as prophylactic use of FLC increases because of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic in sub-Saharan Africa.     

Identification of Candida species isolated from patients in intensive care unit and in vitro susceptibility to fluconazole for a 3-year period

Species level identification of Candida and antifungal susceptibility testing is not generally performed in routine laboratory practice. There is limited information about the distribution of Candida species and antifungal susceptibility in Turkey. In this study, we aimed at identifying Candida isolates to species level from various samples obtained from patients treated in an intensive care unit between 2002 and 2005 and to evaluate fluconazole susceptibilities of the isolates. A total of 320 Candida isolates obtained from 270 patients were identified by conventional methods and using API (Candida and/or 20C AUX) system. Antifungal susceptibility testing was performed by broth microdilution method. Candida albicans was isolated with the highest frequency (65.6%) followed by C. parapsilosis (11.3%), C. glabrata (8.8%) and C. tropicalis (7.8%). Of all the isolates, 92.9% revealed susceptibility to fluconazole. Susceptibility to fluconazole was highest for C. albicans followed by C. parapsilosis and C. glabrata. The MIC(90) values for C. albicans, C. parapsilosis, C. glabrata and C. tropicalis were 1, 2, 8 and 4 mug ml(-1) respectively. Fluconazole remains effective against both C. albicans and the majority of non-albicans Candida species. In this study, we determine the distribution of Candida species and evaluate the susceptibilities of the isolates, particularly for the azoles.     

Susceptibility and trailing growth of Candida albicans to fluconazole: results of a Korean multicentre study

The work reported here is the first nationwide, multicenter surveillance study conducted in Korea to obtain data on fluconazole susceptibility of Candida albicans (C. albicans) isolates. A total of 1137 isolates of C. albicans obtained from 17 university hospitals in South Korea during the 6-month period, July through December 2004, were tested. No resistant strains were observed in any of the isolates. Only five of the 1137 isolates (0.44%) of C. albicans were found to be susceptible dose dependent, with all remaining strains (99.56%) susceptible to fluconazole. Trailing growth at 48 h was found in only four isolates (0.35%).     

Influence of voriconazole and fluconazole on reconstituted multilayered oesophageal epithelium infected by Candida albicans

Reconstituted multilayered oesophageal epithelium appears to be a good basis to test the efficacy of voriconazole (VOR) and fluconazole (FLU) in the tissue. The resulting model of a Candida oesophagitis was approaching the in vivo situation. We infected the tissue with 2 x 10(6) cfu of the Candida albicans strain SC5314. In the trials with FLU we also used clinical strains. Four hours after infection a good growth of C. albicans appeared mainly with hyphae on the surface of the tissue and a tendency to invasion. The destruction of the tissue began after 36 h. VOR (2 and 16 microg ml-1, respectively) prevented the penetration of hyphae into the tissue, when it was given 4-8 h after infection. It was less effective in reduction of Candida growth on the tissue surface. When VOR was given 16-24 h postinfection, the Candida infiltration stopped more slowly. Thirty-six hours after infection VOR application could not stop the destruction of the tissue despite reducing the fungi. The results with FLU (32 microg ml-1) were in principle the same, but not so distinct. FLU seems to be more effective against clinical strains of C. albicans than against the type strain.     

Oropharyngeal carriage of Candida species in HIV-infected patients in India

The present investigation represents the first study of oropharyngeal carriage of Candida and other yeasts in HIV-infected patients in India. One hundred and fifty HIV-positive patients were investigated by culturing their swish samples on plates of CHROMagar Candida. Ninety-eight patients (65.3%) were positive for Candida and four (2.7%) were positive for other yeasts. Among them, the first Indian C. dubliniensis isolate has been recovered. Molecular typing of selected C. albicans isolates by AP-PCR revealed two major genotypes based on the banding patterns. The susceptibilities of 30 Candida isolates to five antifungal agents including the new triazole voriconazole were determined in a micro-dilution test, according to the NCCLS protocol M 27. All the 22 C. albicans isolates were susceptible to five antimycotic agents (flucytosine, amphotericin B, fluconazole, voriconazole and itraconazole) except one isolate (VPCI-122), which was resistant to flucytosine (MIC > or = 64 mg l-1). The azole-resistant isolates reported here endorse the role of antifungal susceptibility testing whenever antifungal treatment with azoles is planned.     

In vitro susceptibility testing of amorolfine in pathogenic fungi isolated from dermatomycosis patients in China

The antifungal susceptibility of isolates from Chinese dermatomycosis patients to amorolfine was investigated following National Committee for Clinical Laboratory Standards (NCCLS) protocols. In total, 383 isolates were tested, including 132 strains from tinea pedis, 148 strains from tinea corporis/cruris, and 103 strains from onychomycosis. The minimum inhibitory concentration (MIC) of amorolfine against dermatophytes ranged from 0.01 to 0.08 microg ml(-1). The MIC(50) and MIC(90) of amorolfine for Trichophyton rubrum were both equal to 0.04 micro ml(-1); for T. mentagrophytes these MICs were 0.04 microg ml(-1) and 0.08 microg ml(-1) respectively; and for Epidermophyton floccosum they were 0.02 microg ml(-1) and 0.04 microg ml(-1) respectively. The MIC range of amorolfine against Candida parapsilosis was 0.5-16 microg ml(-1). MIC(50) and MIC(90) for C. parapsilosis were 0.5 and 2 microg ml(-1). MIC ranges of amorolfine against Scopulariopsis spp. and Acremonium spp. were 0.5-4 and 2-8 microg ml(-1), respectively. Candida albicans, Fusarium solani and Aspergillus flavus required relatively higher concentrations of amorolfine to inhibit their growth (MIC 0.125-64 microg ml(-1), MIC(50) and MIC(90) were 4 and 64 microg ml(-1)). The results demonstrated that amorolfine is the only topical agent that has such a potent antifungal activity and a broad spectrum against a wide range of pathogenic fungi.     

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.     

In vitro activities of new and conventional antimycotics against fluconazole-susceptible and non-susceptible Brazilian Candida spp. isolates

The substantial increase in the rate of azole resistant Candida spp. yeast infections has become a serious treatment problem requiring new and more active antifungal agents. In this study, the in vitro activities of ravuconazole and albaconazole were compared with those of amphotericin B, flucytosine, itraconazole and fluconazole against 162 Brazilian isolates of Candida spp. from which 48 isolates had previously shown lower susceptibility or resistance to fluconazole. Ravuconazole susceptibility ranged from 84.6% (Candida albicans) to 100% for other species and albaconazole MIC(90) was < or =1.0 microg ml(-1) for all the species emphasising the potent activity of these triazoles. To our knowledge this is the first study evaluating the susceptibility of C. dubliniensis to albaconazole.     

Fluconazole susceptibility of Candida spp. isolates collected over nine years in a teaching hospital of Ancona, Italy

The in vitro activity of fluconazole was investigated against 476 yeast isolates collected during a 9-year period (1997-2005) from patients hospitalised in a teaching hospital of Ancona. They included 373 isolates of Candida albicans, 53 of Candida glabrata and 50 of Candida parapsilosis. Minimum inhibitory concentrations (MICs) determined in accordance with the Clinical Laboratory Standards Institute methodology showed that 96% of the isolates were susceptible (MIC < or =8.0 microg/ml). The uncommon, resistant isolates (MIC > or =64 microg/ml) were randomly distributed over time.Our data show that resistance to fluconazole in this geographical area is a rare event and suggest that this triazole can still represent first-line therapy in our institution.     

Azole resistance in neonatal intensive care units in Argentina

The aim of this study was to determine the antifungal susceptibility profile and to detect resistant strains of yeast species isolated from neonates in Intensive Care Units. 92 strains isolated from 25 bloodstream cultures, 20 venous catheters, 23 suprapubic aspirations and 24 rectal swabs were studied. A Candida glabrata strain resistant to fluconazole was detected. Candida krusei appeared with its inherent resistance to fluconazole and showed cross-resistance to itraconazole. Two Candida albicans strains were resistant to azoles, one to itraconazole and the other to fluconazole with a high minimum inhibitory concentration (MIC) for itraconazole. All Candida tropicalis strains were susceptible to fluconazole but two of them showed resistance to itraconazole. The detection of resistant strains in neonates whom had not received previous antifungal therapy is noteworthy. The variations in the epidemiology of fungal infections observed and the antifungal resistance detected emphasize the importance of performing a regular surveillance to observe and to assess them.     

Effect of ketoconazole, itraconazole and fluconazole on the gastrointestinal colonization of mice by Candida albicans

Crl:CD1 (ICR) BR mice were colonized in the gastrointestinal (GI) tract with Candida albicans. This strain was susceptible to ketoconazole (MIC=0.25 microg/ml), itraconazole (minimum inhibitory concentration, MIC=0.25 microg/ml), and fluconazole (MIC=4 microg/ml). Subsequently the animals received monotherapy with ketoconazole by mouth (equivalent to human dose of 2.9 mg/kg/day), or itraconazole by mouth (equivalent to human dose of 2.9 mg/kg/day), or fluconazole either subcutaneously (equivalent to human dose of 2.2 mg/kg/day), or by mouth (equivalent to human dose of 2.2 mg/kg/day), for 10 days. Quantitative stool cultures at the end and one week after the end of treatment revealed that all three azoles caused a small and statistically non significant reduction of C. albicans concentration in the stools. The different route of administration of fluconazole did not produce different results. In conclusion, these azoles, used at the present doses and schedules, have minimal effect on murine GI colonization by this strain of C. albicans which is susceptible but with rather increased MICs.     

In-vitro activity of the synthetic protegrin IB-367 alone and in combination with antifungal agents against clinical isolates of Candida spp

The in vitro activity of the peptide IB-367, alone or combined with either fluconazole (FLU) or amphotericin B (AMB), was investigated against 25 Candida isolates belonging to five species. IB-367 minimum inhibitory concentrations (MICs) ranged from 2.0 to 32 microg/ml and it was active against both FLU-susceptible and - resistant isolates. A rapid fungicidal activity was observed. Synergism was documented in 41.6% and 44% of IB-367/FLU and IB-367/AMB interactions, respectively. Antagonism was never observed. The broad antifungal activity and the positive interactions with AMB and FLU suggest that IB-367 represents a promising candidate against infections due to Candida spp.     

Correlation between phospholipase of Candida albicans and resistance to fluconazole

The objective of this study was to compare phospholipase production between fluconazole-resistant and fluconazole-susceptible strains of Candida albicans in order to explore the relationship between resistance to antifungal drugs and virulence of C. albicans. Fifteen each of fluconazole-resistant (MIC ≥ 64 μg ml(-1)) and fluconazole-susceptible (MIC ≤ 8 μg ml(-1)) strains of C. albicans were incubated on egg yolk agar to detect phospholipase activity. Virulence of C. albicans was assessed by the average survival time of infected mice. Expression of phospholipase B1 mRNA and protein were detected by RT-PCR and Western blot method. Significant differences between the two groups of Candida strains were observed in phospholipase activity and average survival time of infected mice. The expression of phospholipase B1 mRNA and protein (both of secreted and intracellular forms) were higher in resistant strains than in susceptible strains. The results indicate that the phospholipase activity of C. albicans may be related to its resistance to antifungal drugs.     

Antifungal and cancer cell growth inhibitory activities of 1-(3',4',5'-trimethoxyphenyl)-2-nitro-ethylene

The antifungal and cancer cell growth inhibitory activities of 1-(3',4',5'-trimethoxyphenyl)-2-nitro-ethylene (TMPN) were examined. TMPN was fungicidal for the majority of 132 reference strains and clinical isolates tested, including those resistant to fluconazole, ketoconazole, amphotericin B or flucytosine. Minimum fungicidal concentration/minimum inhibitory concentration (MFC/MIC) ratios were < or = 2 for 96% of Cryptococcus neoformans clinical isolates and 71% of Candida albicans clinical isolates. TMPN was fungicidal for a variety of other basidiomycetes, endomycetes and hyphomycetes, and its activity was unaffected by alterations in media pH. The frequency of occurrence of fungal spontaneous mutations to resistance was <10(-6). Kill-curve analyses confirmed the fungicidal action of TMPN, and demonstrated that killing was concentration- and time-dependent. At sub-MIC exposure to TMPN, C. albicans did not exhibit yeast/hyphae switching. TMPN was slightly cytotoxic for murine and human cancer cell lines (GI50=1-4 microg ml(-1)), and weakly inhibited mammalian tubulin polymerization (IC50=0.60 microg ml(-1)).     

Antifungal susceptibility of Candida species isolated from patients with candidemia in southern Taiwan, 2007–2012: impact of new antifungal breakpoints

The Clinical and Laboratory Standard Institute (CLSI) revised the clinical breakpoints (CBPs) for the azoles and echinocandins against Candida species in 2012. We aimed to report the epidemiology of candidemia and antifungal susceptibility of Candida species and evaluate the impact of new CBPs on antifungal susceptibility in our region. All blood isolates of Candida species were obtained from 2007 to 2012. The minimum inhibitory concentrations of fluconazole, voriconazole, echinocandins and flucytosine against Candida isolates were determined by Sensititre YeastOne system. Differences in susceptibility rates between the CBPs of previous and revised versions of CLSI were examined. Of 709 Candida isolates, the fluconazole‐susceptible rate was 96.5% in Candida albicans, 85.8% in Candida tropicalis and 92.1% in Candida parapsilosis by the revised CBPs. Compared with the susceptibility results by previous CBPs, the marked reductions in susceptibility of C. albicans, C. tropicalis and C. parapsilosis to fluconazole, that of C. tropicalis and C. parapsilosis to voriconazole, that of C. tropicalis and Candida glabrata to anidulafungin and that of C. tropicalis, C. glabrata and Candida krusei to caspofungin by revised CBPs were found. In conclusion, Candida albicans and C. parapsilosis remain highly susceptible to fluconazole. The non‐susceptible rates of Candida species to azoles and echinocandins increase with interpretation by the revised CBPs.     

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.