Delivery of Amphotericin B to Candida albicans by Using Biomachined Lab-on-a-Chip

2017 ◽  
Vol 30 ◽  
pp. 24-30 ◽  
Author(s):  
M. Hanif Nadhif ◽  
Yudan Whulanza ◽  
Jos Istiyanto ◽  
Boy M. Bachtiar
Keyword(s):  
Candida Albicans ◽  
Amphotericin B ◽  
Continuous Flow ◽  
Drug Testing ◽  
Lab On A Chip ◽  
Yeast Nitrogen Base ◽  
Mold Surface ◽  
Cnc Milling ◽  
Polydimethyl Siloxane ◽  
Nitrogen Base

This paper investigates the ability of biomachined lab-on-a-chip (LoC) to perform drug testing of Amphotericin B to the Candida albicans. The chip is made of polydimethyl siloxane (PDMS). Molds are patterned using CNC milling followed by biomachining. CNC milling process creates channel features on the bottom mold, while biomachining forms rough surface on the channels. After the molds are created, LoC can be manufactured using those molds. Hence, contour surface on LoC’s channels is also realized following the mold surface. Later, Candida albicans is seeded on the LoC’s channels for 24 and 48 hours with the continuous flow of Yeast Nitrogen Base (YNB) Sterile. Then, cell viability is tested using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium (MTT).The result shows that C. albicans could adhere and grow in the LoC channels. Based on this result, drug testing is conducted in the presence and absence of Amphotericin B (Amp B) under two schemes: without (static) and with (dynamic) the continuous flow of YNB Sterile and Amp B. After 48 hour incubation period, C. albicans biofilm of 28.72 % is shown during dynamic scheme, whereas static scheme had C. albicans biofilm of 99.32 % indicating that the dynamic scheme provides a better efficacy compared to the static scheme.

scholarly journals Flow Cytometry Antifungal Susceptibility Testing of Pathogenic Yeasts other than Candida albicans and Comparison with the NCCLS Broth Microdilution Test

2000 ◽  
Vol 44 (10) ◽  
pp. 2752-2758 ◽  
Author(s):  
Rama Ramani ◽  
Vishnu Chaturvedi
Keyword(s):  
Flow Cytometry ◽  
Candida Albicans ◽  
Amphotericin B ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Antifungal Susceptibility Testing ◽  
Broth Microdilution ◽  
Nitrogen Base ◽  
Microdilution Method ◽  
Broth Microdilution Method

ABSTRACT Candida species other than Candida albicansfrequently cause nosocomial infections in immunocompromised patients. Some of these pathogens have either variable susceptibility patterns or intrinsic resistance against common azoles. The availability of a rapid and reproducible susceptibility-testing method is likely to help in the selection of an appropriate regimen for therapy. A flow cytometry (FC) method was used in the present study for susceptibility testing ofCandida glabrata, Candida guilliermondii,Candida krusei, Candida lusitaniae,Candida parapsilosis, Candida tropicalis, andCryptococcus neoformans based on accumulation of the DNA binding dye propidium iodide (PI). The results were compared with MIC results obtained for amphotericin B and fluconazole using the NCCLS broth microdilution method (M27-A). For FC, the yeast inoculum was prepared spectrophotometrically, the drugs were diluted in either RPMI 1640 or yeast nitrogen base containing 1% dextrose, and yeast samples and drug dilutions were incubated with amphotericin B and fluconazole, respectively, for 4 to 6 h. Sodium deoxycholate and PI were added at the end of incubation, and fluorescence was measured with a FACScan flow cytometer (Becton Dickinson). The lowest drug concentration that showed a 50% increase in mean channel fluorescence compared to that of the growth control was designated the MIC. All tests were repeated once. The MICs obtained by FC for all yeast isolates except C. lusitaniae were in very good agreement (within 1 dilution) of the results of the NCCLS broth microdilution method. Paired ttest values were not statistically significant (P = 0.377 for amphotericin B; P = 0.383 for fluconazole). Exceptionally, C. lusitaniae isolates showed higher MICs (2 dilutions or more) than in the corresponding NCCLS broth microdilution method for amphotericin B. Overall, FC antifungal susceptibility testing provided rapid, reproducible results that were statistically comparable to those obtained with the NCCLS method.

scholarly journals Effect of pH on the In Vitro Activities of Amphotericin B, Itraconazole, and Flucytosine against Aspergillus Isolates

2004 ◽  
Vol 48 (8) ◽  
pp. 3147-3150 ◽  
Author(s):  
D. T. A. Te Dorsthorst ◽  
J. W. Mouton ◽  
C. J. P. van den Beukel ◽  
H. A. L. van der Lee ◽  
J. F. G. M. Meis ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Amphotericin B ◽  
Antifungal Agents ◽  
Poor Correlation ◽  
Yeast Nitrogen Base ◽  
Broth Microdilution ◽  
In Vitro Susceptibility ◽  
Nitrogen Base ◽  
The Poor

ABSTRACT The in vitro susceptibilities of 21 Aspergillus isolates were tested against three antifungal agents in RPMI 1640 and yeast nitrogen base at pH 5.0 and 7.0 by a broth microdilution format of the NCCLS method. The MICs of amphotericin B and itraconazole were higher, while those of flucytosine were lower, at pH 5.0 than at pH 7.0. The poor correlation between in vitro results and clinical outcome could be due to a difference in pH between the in vitro susceptibility test and at the site of infection.

scholarly journals Reversible fluconazole resistance in Candida albicans: a potential in vitro model.

1997 ◽  
Vol 41 (3) ◽  
pp. 535-539 ◽  
Author(s):  
H M Calvet ◽  
M R Yeaman ◽  
S G Filler
Keyword(s):  
Candida Albicans ◽  
Molecular Mechanisms ◽  
Rabbit Model ◽  
Serial Passage ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Fluconazole Resistance ◽  
Karyotypic Analysis

To study the development and potential mechanisms of antifungal resistance in relation to antifungal exposure, reversible fluconazole resistance was examined in vitro. Candida albicans ATCC 36082 blastospores were passed in liquid yeast nitrogen base medium containing either 4, 8, 16, or 128 micrograms of fluconazole per ml, and susceptibility testing was performed after each passage. High-level fluconazole resistance (50% inhibitory concentration, > 256 micrograms/ml) developed in the isolates after serial passage in medium containing 8, 16, or 128 micrograms of fluconazole per ml, but not in isolates passed in 4 micrograms of fluconazole per ml. Reduced susceptibility was noted within four to seven passages, which was equivalent to 14 to 19 days of exposure to the drug. However, all isolates returned to the susceptible phenotype after 8 to 15 passages in medium lacking the drug; thus, fluconazole resistance was reversible in vitro. In vivo, organisms retained the resistant phenotype after a single passage in the rabbit model of infective endocarditis. Restriction digest profiles and karyotypic analysis of the parent strain and selected fluconazole-resistant and -susceptible isolates from each group were identical. Investigations into the molecular mechanisms of this reversible resistance failed to reveal increased accumulation of mRNA for 14 alpha-demethylase, the target enzyme for fluconazole, or for the candidal multidrug transporters CDR1 and BENr. This process of continuous in vitro exposure to antifungal drug may be useful as a model for studying the effects of different antifungal agents and dosing regimens on the development of resistance and for defining the mechanism(s) of reversible resistance.

scholarly journals Detection of Resistance to Amphotericin B amongCryptococcus neoformans Clinical Isolates: Performances of Three Different Media Assessed by Using E-Test and National Committee for Clinical Laboratory Standards M27-A Methodologies

1998 ◽  
Vol 36 (10) ◽  
pp. 2817-2822 ◽  
Author(s):  
M. Lozano-Chiu ◽  
V. L. Paetznick ◽  
M. A. Ghannoum ◽  
J. H. Rex
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Diffusion Method ◽  
National Committee ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Agar Diffusion ◽  
Agar Diffusion Method

Although reliable detection of resistance in vitro is critical to the overall performance of any susceptibility testing method, the recently released National Committee for Clinical Laboratory Standards M27-A methodology for susceptibility testing of yeasts discriminates poorly between resistant and susceptible isolates ofCandida spp. We have previously shown that both substitution of antibiotic medium 3 for RPMI 1640 medium in the microdilution variant of the M27-A method and use of the E-test agar diffusion methodology permit detection of amphotericin B-resistantCandida isolates. To determine the relevance of these observations to Cryptococcus neoformans, we have evaluated the performances of both the M27-A and the E-test methodologies with this yeast using three different media (RPMI 1640 medium, antibiotic medium 3, and yeast nitrogen base). As with Candida, we found that only antibiotic medium 3 permitted consistent detection of resistant isolates when testing was performed in broth by the M27-A method. When testing was performed by the E-test agar diffusion method, both RPMI 1640 medium and antibiotic medium 3 agar permitted ready detection of the resistant isolates. Reading of the results after 48 h of incubation was required for testing in broth by the M27-A method, while the MIC could be determined after either 48 or 72 h when the agar diffusion method was used.

scholarly journals Results Obtained with Various Antifungal Susceptibility Testing Methods Do Not Predict Early Clinical Outcome in Patients with Cryptococcosis

2006 ◽  
Vol 50 (7) ◽  
pp. 2464-2470 ◽  
Author(s):  
E. Dannaoui ◽  
M. Abdul ◽  
M. Arpin ◽  
A. Michel-Nguyen ◽  
M. A. Piens ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Amphotericin B ◽  
Antifungal Susceptibility ◽  
Yeast Nitrogen Base ◽  
Broth Microdilution ◽  
Nitrogen Base ◽  
First Line Therapy ◽  
Multicenter Prospective Study ◽  
In Vitro Antifungal Susceptibility

ABSTRACT The in vitro susceptibilities of Cryptococcus neoformans isolates from consecutive human immunodeficiency virus-positive and -negative patients to the antifungal agents fluconazole, amphotericin B, and flucytosine were determined by different techniques, including the CLSI method, Etest, and broth microdilution in yeast nitrogen base (YNB) medium, during a multicenter prospective study in France. The relationship between the in vitro data and the clinical outcome 2 weeks after the initiation of antifungal therapy was assessed. In addition, the correlation between the strain serotype and the in vitro activities of the antifungals was determined, and the susceptibility results obtained with the different techniques were also compared. Thirty-seven patients received a combination of amphotericin B with flucytosine as first-line therapy, 22 were treated with amphotericin B alone, and 15 received fluconazole alone. Whatever the antifungal tested, there was no trend toward higher MICs for strains isolated from patients who failed to respond to a given therapy compared to those from patients who did not with either the CLSI method, Etest, or broth microdilution in YNB medium. The MICs obtained by the CLSI or Etest method were significantly lower for serotype D strains than for serotype A strains for both fluconazole and amphotericin B, while flucytosine MICs were not different according to serotype. These findings suggest that the in vitro antifungal susceptibility of C. neoformans, as determined with the techniques used, is not able to predict the early clinical outcome in patients with cryptococcosis.

scholarly journals Neonatal sepsis due to non-albicans Candida species and their susceptibility to antifungal agents: first report from Bangladesh

2021 ◽  
Vol 14 (2) ◽  
pp. 19-26
Author(s):  
Rafia Afreen Jalil ◽  
KM Shahidul Islam ◽  
Lovely Barai ◽  
Shahida Akhter
Keyword(s):  
Amphotericin B ◽  
Neonatal Sepsis ◽  
Candida Species ◽  
Antifungal Agents ◽  
Tertiary Care ◽  
Yeast Nitrogen Base ◽  
Dhaka City ◽  
Suspected Sepsis ◽  
Nitrogen Base ◽  
Broth Microdilution Method

Background and objectives: Frequency of neonatal sepsis in Neonatal Intensive Care Units (NICU) has been increasing worldwide over the last decades. The emergence of non-albicans Candida (NAC) species and their resistance to common antifungal agents become an important preventive and therapeutic issue. The present study was undertaken to find out the role of NAC species in neonatal sepsis/candidemia in the NICUs of hospitals of Dhaka city. The susceptibility pattern of NAC species to antifungal agents was also determined. Materials and methods: Suspected cases of neonatal sepsis admitted in NICU of four tertiary care hospitals of Dhaka city, from March to December 2018 were enrolled. In this cross sectional study, blood samples were collected from neonates with suspected sepsis for culture. Identification of Candida species was done by carbohydrate (CHO) assimilation tests using swab auxanographic technique, CHO impregnated yeast nitrogen base plate method (YNB), microtiter plate based miniaturized method and by HiCromeTM Candida Differential Media. Susceptibility of the isolated Candida species to antifungal agents was determined by disk diffusion (DD) and by minimum inhibitory concentration (MIC) methods. MIC was determined by broth microdilution method using RPMI 1640 and trypticase soy broth (TSB). Results: In the present study, NAC species were isolated from 39.7% neonates. C. tropicalis was the predominant species (81.0%) followed by C. parapsilosis (12.1%), C. auris (5.2%) and C. dubliniensis (1.7%). Isolated NAC species were 98.3% sensitive to voriconazole. Sensitivity to fluconazole, ketoconazole, itraconazole, and clotrimazole was 3.5%, 15.5%, 86.2% and 56.9% respectively by DD method. All the isolates (100%) were sensitive to miconazole and nystatin. All the C. tropicalis, C. auris and C. dubliniensis were sensitive to amphotericin B and anidulafungin. One and four C. parapsilosis were found resistant to amphotericin B and anidulafungin respectively. The MIC results obtained by using RPMI 1640 and TSB as growth medium were concordant suggesting that TSB media was a good alternative to expensive RPMI 1640. Conclusion: The advent of NAC species merits attention as they are highly resistant to most of the azoles. Therefore, speciation of Candida in neonatal candidemia is essential to institute appropriate antifungal therapy. Ibrahim Med. Coll. J. 2020; 14(2): 19-26

scholarly journals Counter-Acting Candida albicans-Staphylococcus aureus Mixed Biofilm on Titanium Implants Using Microbial Biosurfactants

Polymers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 2420
Author(s):  
Erica Tambone ◽  
Alice Marchetti ◽  
Chiara Ceresa ◽  
Federico Piccoli ◽  
Adriano Anesi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Metabolic Activity ◽  
Titanium Implants ◽  
Microbial Colonization ◽  
Total Biomass ◽  
Yeast Nitrogen Base ◽  
Biofilm Inhibition ◽  
Nitrogen Base ◽  
Primary Osteoblasts

This study aimed to grow a fungal-bacterial mixed biofilm on medical-grade titanium and assess the ability of the biosurfactant R89 (R89BS) coating to inhibit biofilm formation. Coated titanium discs (TDs) were obtained by physical absorption of R89BS. Candida albicans-Staphylococcus aureus biofilm on TDs was grown in Yeast Nitrogen Base, supplemented with dextrose and fetal bovine serum, renewing growth medium every 24 h and incubating at 37 °C under agitation. The anti-biofilm activity was evaluated by quantifying total biomass, microbial metabolic activity and microbial viability at 24, 48, and 72 h on coated and uncoated TDs. Scanning electron microscopy was used to evaluate biofilm architecture. R89BS cytotoxicity on human primary osteoblasts was assayed on solutions at concentrations from 0 to 200 μg/mL and using eluates from coated TDs. Mixed biofilm was significantly inhibited by R89BS coating, with similar effects on biofilm biomass, cell metabolic activity and cell viability. A biofilm inhibition >90% was observed at 24 h. A lower but significant inhibition was still present at 48 h of incubation. Viability tests on primary osteoblasts showed no cytotoxicity of coated TDs. R89BS coating was effective in reducing C. albicans-S. aureus mixed biofilm on titanium surfaces and is a promising strategy to prevent dental implants microbial colonization.

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