Isolation, identification and antifungal susceptibility of Candida in patients with fungal sepsis

Background: In this study, our aim was to identify and isolate Candida species from patients admitted in ICU,s of our hospital and to determine their susceptibilities to various antifungal agents so as to find the local resistance pattern and guide for empirical treatment.Methods: In our study 37 strains of candida were isolated (4 Candida albicans, 33 Non-albicans Candida strains). Candida species were identified by conventional, biochemical and molecular methods. Antifungal susceptibility tests for amphotericin B, fluconazole, itraconazole, ketoconazole and voriconazole were performed with broth microdilution method and E- tests as described by National Committee for Clinical Laboratory Standards (NCCLS).Results: Out of 37 Candida strains, the most prevalent species were C. tropicalis (43.2%), C. parapsilosis (24.3%), C. krusei (16.2%), C. albicans (10.8%), and C. glabrata (2.7%). Among all strains four strains (10.8 %) were resistant, two Candida albicans where found resistant to fluconazole one Candida krusei and one Candida parapsilosis were found to be resistant to all azoles.Conclusions: Candidemia continues to be associated with substantial morbidity and mortality and non albicans Candida species are the commonly isolated pathogen from those patients admitted in tertiary care hospitals in Indian scenario. Thus, it is imperative to perform antifungal susceptibility to select appropriate and effective antifungal therapy.

Deciphering polymyxin B minimum inhibitory concentration from colistin minimum inhibitory concentration and vice versa: An analysis on 156 carbapenem-resistant Enterobacteriaceae isolates

The susceptibility determination to polymyxins (colistin and polymyxin B) remains a challenge for clinical microbiology laboratories. We evaluated the minimum inhibitory concentration (MIC) of both antimicrobials by the broth microdilution method in a selected subset of 156 carbapenem-resistant Enterobacteriaceae (CRE) isolates. Good concordance between polymyxin B and colistin MIC values was obtained, and there was 98% categorical agreement in CRE isolates. Future large-scale multicentre study is needed to draw conclusion if the MIC of colistin can be used to extrapolate the MIC of polymyxin B and vice versa.

Evaluation of the capabilities of disc diffusion and Vitek2-compact GN13 methods for testing antimi-crobial susceptibility and screening of ESBLs Enterobacteriaceae clinical isolates / 中华微生物学和免疫学杂志

Objective To evaluate the capabilities of disc diffusion and Vitek2-compact GN13 methods for testing antimicrobial susceptibility of screening ESBLs ( extended-spectrumβ-lactamase) in En-terobacteriaceae clinical isolates.Methods A total of 93 Enterobacteriaceae strains were isolated from pa-tients with intra-abdominal infections in 21 hospitals during 2011 to 2012.The in vitro minimum inhibition concentration ( MIC ) values of ampicillin-sulbactam, piperacillin-tazobactam, ertapenem, ceftazidime, ceftriaxone, cefepime, imipenem, amikacin, ciprofloxacin and levofloxacin were determined by disc diffu-sion, Vitek2-compact GN13 and broth microdilution methods, respectively.Categorical agreement ( CA ) rates of disc diffusion and Vitek2-compact GN13 methods were determined by using broth microdilution meth-od as the reference method.The genes encoding ESBLs were screened in Escherichia coli (E.coli), Kleb-siella pneumoniae (K.pneumonia), Klebsiella oxytoca (K.oxytoca) and Proteus mirabilis (P.mirabilis) strains by using PCR analysis and gene sequencing.Disc diffusion and Vitek2-compact GN13 methods were used for the phenotypic confirmatory test of ESBLs and the sensitivity, specificity, positive predictive value and negative predictive value of the two tests were evaluated.Results The CA values of disc diffusion and Vitek2-compact GN13 methods for the 10 antibiotics were all >90% as compared with broth microdilution method.The major error (ME) rate for ertapenem was 3.2%and the very major error (VME) rates for am- picillin-sulbactam, ceftazidime and cefepime tests were all 2.2% by using Vitek2-compact GN13 method. The sensitivity, specificity, positive predictive value and negative predictive value of disc diffusion and Vitek2-compact GN13 methods in the phenotypic confirmatory test of ESBLs were 96.7%(29/30), 100%(20/20), 100%(30/30) and 95%(19/20), respectively.Conclusion Both disc diffusion and Vitek2-compact GN13 methods could be used for testing the antimicrobial susceptibility and the detection of ESBLs in Enterobacteriaceae clinical isolates with the advantage of accuarcy.Attention should be paid to the posibil-lity of oaurance of ME and VME when testing ertapenem, ampicillin-sulbactam, ceftazidime and cefepime by using Vitek2-compact GN13 method.

Study on disk diffusion method for antimicrobial susceptibility test of Candida isolated from patients with lung cancer / 肿瘤研究与临床

Objective To compare the ROSCO disk diffusion method with broth microdilution method (CLSI, M27-A) for antimicrobial susceptibility test of Candida species isolated from patients with lung cancer. Methods Danish ROSCO company disk diffusion testing method and bio Merieux ATB FUNGUR2 were applied to test 5-flucytosine, fluconazole, itraconazole and amphotericin B antimicrobial susceptibility for 78 Candida species strains isolated from patients with lung cancer. Results Through evaluating the susceptibility to 5-flucytosine, amphotericin B, fluconazole and itraconazole by disk diffusion method, the Kappa value was 0.89. The sensitive strains detected by one method did not show resistance in another method. The sensitive rates of 78 strains of Candida species to 5-flucytosine, amphotericin B, fluconazole and itraconazole were 88.20 %, 89.17 %, 56.34 % and 52.12 %. The susceptibility of C.albicans, C.tropicalis, C.glabrata and C.krusei to four kinds of antifungal agents was 90.95 %, 85.71 %, 67.50 % and 41.67 %respectively. Conclusions Results of disk diffusion method coincide well with broth microdilution method. It can be chosen as a clinical routine method for antimicrobial susceptibility test.

Actividad antifúngica de extractos de biomasa celular de neem sobre aislamientos de dermatofitos / Antifungal activity of neem cellular biomass extracts on dermatophytes isolates

Las líneas celulares de neem (Azadirachta indica A. Juss.) cultivadas en suspensión líquida han demostrado producir metabolitos secundarios bioactivos, particularmente triterpenoides. En consecuencia, se han realizado estudios para el control de microorganismos de importancia médica, como los hongos dermatofitos. El objetivo principal de este trabajo fue evaluar a través de un método de referencia in vitro la actividad antifúngica de diferentes extractos de cultivos celulares de neem sobre varios aislamientos de Trichophyton mentagrophytes, Trichophyton rubrum y Epidermophyton floccosum. Se realizó un escalado de cultivos de suspensiones celulares de neem, a partir de los cuales se obtuvo un extracto crudo metanólico. Éste extracto fue fraccionado posteriormente por cromatografía en columna de silica gel. Con los extractos obtenidos se determinó la Concentración Mínima Inhibitoria (CMI), siguiendo el método de microdilución en caldo M38-2, con cinco aislamientos de T. mentagrophytes, cinco de T. rubrum y tres de E. floccosum. Se usó como control positivo el antimicótico Terbinafina. Los resultados mostraron que el extracto crudo de biomasa celular de neem inhibe el crecimiento hasta en 100 % de T. mentagrophytes, T. rubrum y E. floccosum. Al evaluar las fracciones por separado, se observó que las de menor polaridad exhibieron en general mayor actividad antifúngica (CMI=109 μg/mL) que el extracto crudo per se (CMI=2500 μg/ mL) y las fracciones más polares (CMI=7000 μg/mL). Lo anterior indica que las células de neem cultivadas en suspensión producen compuestos con actividad antifúngica, siendo más bioactivos los presentes en las fracciones de menor polaridad.

Cell lines of neem (Azadirachta indica A. Juss.) grown in liquid suspension have shown to produce bioactive secondary metabolites, particularly triterpenoids. In consequence, its use as a control of medical microorganisms (like dermatophytes) is proposed. The main goal of this study was to assess the antifungal activity of methanolic extracts from neem cultured cell suspensions on several isolates of Trichophyton mentagrophytes (five isolates), Trichophyton rubrum (five isolates) and Epidermophyton floccosum (three isolates). Neem cell suspension cultures were scaled up, from which a raw methanolic extract was obtained. This extract was fractionated by silica gel column chromatography. The raw methanolic extract and its fractions were used in order to determine the Minimal Inhibitory Concentration (MIC) on the dermatophytes isolates by following M38-A2 broth microdilution method. Antimycotic Terbinafine was used as positive control. The results shown that neem raw cellular biomass extract inhibits the growth of T. mentagrophytes, T. rubrum and E. floccosum in at least 100%. In the evaluations of the separated fractions, it was observed that the low polarity fractions had higher antifungal activity (MIC=109 μg/mL) than the raw extract per se (MIC=2500 μg/mL) and the most polar ones (MIC=7000 μg/mL). The latter suggest that neem cells cultured in liquid suspension produces compounds with antifungal activity, being more active those present in the low polarity fractions.

Study of the anti-MRSA activity of Rhizoma coptidis by chemical fingerprinting and broth microdilution methods / 中国天然药物

AIM@#Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium that causes both hospital- and community-acquired infections, and for which single-drug treatments are becoming less efficient. Rhizoma coptidis has been used for more than two thousand years in China to treat diarrhea, fever, and jaundice. In this study, the anti-MRSA activity of Rhizoma coptidis is examined and its effective components sought.@*METHODS@#The mecA and norA genes were determined by PCR amplification and sequencing. Drug susceptibility of Staphylococcus aureus ATCC43300 was performed using the VITEK2 compact system. The chemical fingerprint of Rhizoma coptidis was investigated using HPLC and preparative liquid chromatography, and the anti-MRSA activity was determined using an improved broth microdilution method.@*RESULTS@#The drug susceptibility test revealed that the penicillin-binding protein phenotype of the strain changed in comparison to penicillin-sensitive Staphylococcus aureus. Ten batches of Rhizoma coptidis showed anti-MRSA activity on the norA-negative Staphylococcus aureus strain, as well as the strain that contained a norA gene. The spectrum-effect relationship revealed that the berberine alkaloids were the effective components, within which berberine, coptisine, palmatine, epiberberine, and jatrorrhizine were the major components.@*CONCLUSION@#This study lays a foundation for in vivo studies of Rhizoma coptidis and for the development of multi-component drugs.

Comparison of broth microdilution and agar dilution methods for antifungal susceptibility testing of Malassezia speciesin vitro / 中华皮肤科杂志

Objective To compare broth microdilution and agar dilution methods for in vitro testing of activities of fluconazole,ketoconazole and itraconazole against clinical Malassezia isolates.Methods Broth microdilution and agar dilution methods were used to determine the minimal inhibitory concentration(MIC)of fluconazole,ketoconazole and itraconazole for 27 clinical strains(5 species)of Malassezia.Results The minimal inhibitory concentration(MIC)ranges of fluconazole,ketoconazole and itraconazole were 0.25-≥64 mg/L,≤0.03-0.5 mg/L and ≤0.03-0.125 mg/L respectively as shown by broth microdilution method,2-≥64 mg/L,≤0.03-0.5 mg/L and ≤0.03-0.25 mg/L respectively as revealed by agar dilution method.Both methods demonstrated that itraconazole possessed the strongest activity against Malassezia species,followed by ketoconazole and fluconazole.The agreement rate in MICs between the two methods was 78.8％,85.2％ and 88.9％,respectively for fluconazole,ketoconazole and itraconazole,with the intraclass correlation coefficients (ICCs)being 0.88,0.80 and 0.76 respectively.Conclusions Fluconazole,ketoconazole and itraconazole are highly active against Malassezia species in vitro,and itraconazole is the most active.Broth microdilution and agar dilution method coincide well in,and are applicable for,the antifungal susceptibility testing of Malassezia species in vitro.

In vitro susceptibility testing of dermatophytes isolated in Goiania, Brazil, against five antifungal agents by broth microdilution method / Teste de suscetibilidade in vitro de dermatófitos isolados em Goiânia, Brasil, contra cinco agentes antifúngicos pelo método de microdiluição em caldo

The antifungal activities of fluconazole, itraconazole, ketoconazole, terbinafine and griseofulvin were tested by broth microdilution technique, against 60 dermatophytes isolated from nail or skin specimens from Goiania city patients, Brazil. In this study, the microtiter plates were incubated at 28 ºC allowing a reading of the minimal inhibitory concentration (MIC) after four days of incubation for Trichophyton mentagrophytes and five days for T. rubrum and Microsporum canis. Most of the dermatophytes had uniform patterns of susceptibility to the antifungal agents tested. Low MIC values as 0.03 µg/mL were found for 33.3 percent, 31.6 percent and 15 percent of isolates for itraconazole, ketoconazole and terbinafine, respectively.

Atividades antifúngicas de fluconazol, itraconazol, cetoconazol, terbinafina e griseofulvina foram testadas pelo método de microdiluição em caldo contra 60 isolados de dermatófitos. Os resultados mostraram que todos os isolados produziram crescimento claramente detectável a 28 ºC e a concentração inibitória mínima (CIM) foi determinada após quatro dias de incubação para Trichophyton mentagrophytes e cinco dias para T. rubrum e Microsporum canis. A maioria dos isolados teve um padrão uniforme de suscetibilidade para os agentes antifúngicos testados. Baixos valores de CIM como 0,03 µg/mL foram encontrados para 33,3 por cento, 31,6 por cento e 15 por cento dos isolados para itraconazol, cetoconazol e terbinafina, respectivamente.

The Optimal Wavelength of Spectrophotometric Broth Microdilution Antifungal Susceptibility Test for Flucytosine and Three Azoles / 대한진단검사의학회지

BACKGROUND: There is no guideline for the appropriate wavelength at which to measure the optical density (OD) value in broth microdilution antifungal susceptibility testing, although a spectrophotometric reading method is commonly used. The present study aimed to analyze the difference in the OD values over the range of visible light and to ascertain the optimal wavelength for the spectrophotometric method of microdilution testing. METHODS: We measured the OD of background blank controls of broth medium, antifungal agents, and inocula of five type strains using a Synergy HT multi-detection microplate reader at 5-nm intervals from 380 nm to 760 nm. We also estimated the OD differences between the 50% of growth control and blank control. RESULTS: The OD of the blank control showed a parabola shape with two peaks and steadily decreased at longer wavelengths. The curves of the antifungal agent were similar to those of blank controls, and the influence of each antifungal agent on the OD was minimal. For the difference in OD between 50% of growth control and the blank control, the curve was the opposite of the blank control, and the OD increased steadily at the wavelengths above 600 nm. CONCLUSIONS: The range between 600 nm and 700 nm was the optimal wavelength for broth microdilution antifungal susceptibility testing, although any wavelength within the visible light spectrum can be used.

Evaluation of Spectrophotometric Broth Microdilution Methods for Determining Antifungal Susceptibilities of Aspergillus Species / 대한진단검사의학회지

BACKGROUND: A spectrophotometric approach to minimum inhibitory concentration (MIC) determination for filamentous fungi may provide an objective and rapid MIC reading, and quantify the hyphal growth of molds. In this study, we evaluated two spectrophotometric broth microdilution methods (SBM) to determine amphotericin B and itraconazole MICs for Aspergillus species isolated from clinical specimens. METHODS: A total of 80 clinical isolates (20 A. fumigatus, 20 A. flavus, 18 A. niger, 20 A. terreus, and 2 A. nidulans) were tested for amphotericin B and itraconazole susceptibility by the broth microdilution method. The MIC endpoint was calculated by the spectrophotometer with microplate reader (SBM-Spec method) or colorimetric XTT (tetrazolium dye) method (SBM-XTT method). The results of the SBM method were compared with those of NCCLS M38-A broth microdilution method. RESULTS: The MICs of amphotericin B by the NCCLS M38-A method ranged from 0.125 to 8 g/mL, and those of itraconazole ranged from 0.25 to 2micrograms/mL. The agreement of SBM-Spec and SBM-XTT methods within one dilution of the NCCLS M38 reference were 98.8% and 96.3% for the ampho-tericin B, and 98.8% and 100% for itraconazole, respectively. The agreements between SBM-Spec and SBM-XTT methods were 97.5% for amphotericin B and 98.8% for itraconazole. CONCLUSIONS: In antifungal susceptibility testing of Aspergillus species, the SBM method includ-ing SBM-Spec and SBM-XTT methods showed high levels of agreements with the NCCLS M38-A method. The SBM methods can be useful in the clinical laboratory.

The Evaluation of Clinical Utility of ATB FUNGUS 2 for Antifungal Susceptibility Testing in Candida Species / 대한임상미생물학회지

BACKGROUND: Although the National Committee for Clinical Laboratory Standards (NCCLS) defined a standard reference broth microdilution method for testing the susceptibility of Candida species to antifungal drugs, many clinical laboratories require easier but reliable alternatives for routine antifungal susceptibility testing. We evaluated ATB FUNGUS 2 (bioMerieux, France.; ATB) compared to the method recommended by the NCCLS (NCCLS). METHODS: A total of 28 strains of Candida species consecutively isolated from blood and CSF cultures at Asan Medical Center from April to June 2004 were tested. In addition, 12 strains comprising C. krusei (3), C. glabrata (7) and C. guilliermondii (2) from the collection of Chonnam National University Hospital were included in the study. These strains were tested for minimum inhibitory concentrations (MICs) against flucytosine (FC), fluconazole (FZ), itraconazole (IZ) and amphotericin B (AB) by both of ATB and NCCLS. In NCCLS, MICs were read using a spectrophotometer after 24 and 48 hour-incubation. RESULTS: The concordance rates of MICs between ATB and NCCLS after 24 hour-incubation were 100%, 75%, 89% and 96% within two-fold dilution and 100%, 97%, 97%, 100% within four-fold dilution for FC, FZ, IZ and AB, respectively. For C. krusei, all three FC and FZ-resistant strains were either intermediate or SDD and one IZ-resistant strain was SDD in ATB, respectively. One C. tropicalis strain resulted in AB MICs of 0.5 microgram/mL in NCCLS, but 2 microgram/mL in ATB. CONCLUSIONS: ATB showed good concordance rates with NCCLS after 24 hour-incubation. ATB appears to be a useful alternatives to NCCLS for routine antifungal susceptibility tests. However, ATB needs further evaluation with more clinical strains, especially those resistant to antifungal agents.

Evaluation of a Spectrophotometric Broth Microdilution Method for Determining Fluconazole Susceptibility of Candida albicans: Influence of RPMI and RPMI-2% Glucose on the Selection of Endpoint Criteria / 대한진단검사의학회지

BACKGROUND: Minimum inhibitory concentration (MIC) endpoint determination is the major varia-tion source for the fluconazole susceptibility test, especially for Candida albicans. In this study, we evaluated spectrophotometric broth microdilution methods using RPMI 1640 and RPMI supple-mented with 18 g of glucose per liter (RPMI-2% glucose) for determining fluconazole susceptibility of C. albicans. METHODS: A total of 129 clinical isolates of C. albicans were tested by the broth microdilution method using RPMI and RPMI-2% glucose. The MIC endpoint was calculated objectively with the spectrophotometer set at 405 nm. These results were compared to those by the National Commit-tee for Clinical Laboratory Standards (NCCLS) macrodilution method and the agar dilution method. RESULTS: The mean absorbances in the drug-free wells in RPMI and RPMI-2% glucose were 0.208 +/- 0.014 and 0.316 +/- 0.061, respectively, at 24 h and 0.339 +/- 0.094 and 0.530 +/- 0.104, respectively, at 48 h (P < 0.01). The agreement of the microdilution method with the RPMI within two doubling dilutions of the macrodilution reference were 91.5% (118/129) at 24 h and 76.7% (99/129) at 48 h. The percentage of agreement in the microdilution method with the RPMI-2% glucose were significantly higher: 100% (129/129) at 24 h and 99.2% (128/129) at 48 h (P < 0.01). In addition, the MIC endpoints were easier to detect in RPMI-2% glucose, because of the greater difference in absorbance in between grown wells and fluconazole-inhibited wells (P < 0.01). CONCLUSIONS: The spectrophotometric microdilution method with RPMI-2% glucose may have an excellent agreement with the NCCLS broth macrodilution method and may provide more easily determined MIC endpoints for fluconazole susceptibility testing for C. albicans.

Amphotericin B and Fluconazole Susceptibility Test of Candida Species: Comparison of Broth Microdilution Method and Agar Dilution Method / 대한임상병리학회지

BACKGROUND: The rising incidence of fungal infections and the increasingly frequent use of antifungal agents have intensified the need for practical and reliable antifungal susceptibility test methods. In the present study, the minimal inhibitory concentration(MIC) distribution of Candida species was investigated and the agar dilution method was compared with the National Committee for Clinical Laboratory Standards(NCCLS) "reference" broth microdilution method for antifungal susceptibility testing. METHODS: A total of 116 clinical isolates of Candida species from patients at Hanyang University Kuri Hospital were studied from October 1997 to July 1999, and the MICs of Candida species were evaluated against amphotericin B and fluconazole by the NCCLS method and the agar dilution method. RESULTS: There were no differences in the MIC50 and MIC90 of Candida albicans and Candida tropicalis against amphotericin B between the two methods, but the MIC50 of C. albicans against fluconazole was higher in the agar dilution method than in the broth microdilution method. The resistant rate of C. albicans against fluconazole was 20.8% in the broth microdilution method and 33.8% in the agar dilution method. For C. tropicalis, 31.3% were resistant to fluconazole in the broth microdilution method and 25.0% in the agar dilution method. The agreement of C. albicans and C. tropicalis between the agar dulution method and the broth microdilution method within one doubling dilution of the microdilution reference were 88.8% for amphotericin B and 34.4% for fluconazole. CONCLUSIONS: The MICs of amphotericin B showed good agreement between the agar dilution and the broth microdilution method. Therefore, resistant strains could easily be detected by screening with agar of 2 g/mL concentrations. However, in the case of fluconazole, the agreement between the two methods was low and the trailing effect could not be ruled out in agar dilution method with some strains, indicating the need for further studies in this area.