Stability and duration of antifungal effects of Nystatin and Fluconazole mixed ...
Original article
J Res Dent Sci
Spring 2014;11(1): 20-25
Stability and duration of antifungal effects of Nystatin and Fluconazole mixed with a tissue conditioner on colonization of Candida
Albicans (in vitro)
Abbas Fallah-tafti1, Abbas Ali jafari2, Leila Mirzaeiipoor3, Hasan Ashoori4
1
Assistant Professor, Prosthodontics Dept, School of Dentistry, Shahid Sadoughi University of Yazd Medical
Sciences, Yazd, Iran.
2
Associate Professor, Basic Science Dept, International Branch, Shahid Sadoughi university of Yazd Medical
Sciences, Yazd, Iran.
3
Dentistry students, School of Dentistry, Shahid Sadoughi university of Yazd Medical Sciences, Yazd, Iran.
4
Laboratory Student, Student Research Committee, Paramedical student, Shahid Sadoughi University of Yazd
Medical Sciences, Yazd, Iran.
Received: Aug 2013
Accepted: Jan 2014
ABSTRCAT
Background and Aim: Tissue conditioners are used to manage irritation of denture bearing mucosa. These
liners, however, promote adherence and colonization by Candida Albicans which can be prevented by incorporating antifungal agents into them. The aim of this in vitro study was to assess the stability and duration of antifungal effects of nystatin and fluconazole mixed with a tissue conditioner on colonization of Candida Albicans.
Materials and Methods:In this experimental study, antifungal test disks were prepared using tissue conditioner
mixed with nystatin and fluconazole. Similar drug free tissue conditioner disks were prepared as controls. The
disks were immersed and kept in artificial sterile saliva until used for susceptibility test. They were placed on the
Sabouraud dextrose agar plates streaked with C. Albicans inoculum and using the agar diffusion test, colonization inhibitory effects were evaluated in one, two, three and 5 day intervals based on saliva immersion period.
The mean inhibitory zone around disks at different time intervals were analyzed with SPSS15 software using
ANOVA and Tukey’s statistical tests for multiple and pairwise comparisons respectively.
Results: The average diameter of inhibitory zone around nystatin and fluconazole disks after 24hour of immersion in saliva were 11.2 and 8.6 mm respectively. The difference in the mean zone of inhibition was statistically
significant after 24 hours (p=0.0001) and 48 hours (p=0.013). This difference was not statistically significant
after 72hours or 5 day interval. . (P>0.05)
Conclusion: The stability of nystatin and fluconazole mixed with a tissue conditioner is not adequate and antifungal effects are seen only for 72 hours while in contact with saliva. Optimum antifungal activity could be
expected in the first 24 hours. Nystatin exhibits higher inhibitory effects compared to fluconazole.
Key words: Enterococcus Faecalis; Rootcanal medicaments; Canalirrigants
INTRODUCTION
The normal flora of human oral cavity comprises various bacterial and fungal microorgan
Corresponding author: Abbas Ali Jafari, Basic
science department, International Campus, 5th Kilometer of Bafgh road, Shahid Sadoughi University of Medical Sciences, Tel: 091333519212 Email: Yazd Jafariabbas@ssu.ac.ir
isms which can transform to pathogenic forms
as a result of denture use 1. The most common
microorganism found in the oral cavity is Candida Albicans which is known to accumulate on
tissue bearing surface of the denture and lead to
Denture Stomatitis (DS) 2. Several predisposing factors have been recognized for oral Candida infection which include: mucosal trauma
due to poor denture adaptation, smoking,
20/Vol 11,No 1 Spring 2014
J Res Dent Sci, Spring 2014
Published by Islamic Azad University , Dental Branch, Tehran , Iran (http://www.jrds.ir)
A.Fallah-tafti, et al.
use of antibiotics, cancer and endocrinological
disorders 3, 4.
Chronic atrophic candidiasis, known as Denture
Stomatitis, is a form of oral candidiasis seen on
palatal and alveolar mucosa of denture wearers. Immunocompromised or diabetic elderly
with complete dentures and poor oral hygiene
are particularly prone to further serous systemic
infections caused by chronic atrophic candidiasis5. Daily disinfection as well as removal of the
appliance at night and use of antifungal mouth
rinses have been proposed for prevention and
treatment of DS 6, 7 A thin layer of tissue conditioner may be used as a buffer for prevention
of trauma in cases where removal of denture is
unlikely 8.
Although the use of tissue conditioners may improve the overall adaption of an ill-fitting denture, they can promote adherence and colonization by Candida Albicans and thus, intensifying
denture related complications 8, 9.
The use of topical antifungal agents and correction of the denture imperfections are recommended to overcome these problems. However,
maintaining an efficient dose of topical antifungals in the oral cavity is difficult. These agents
fail to adhere and remain in contact with oral
mucosal tissue due to regular ingestion and
constant wash out by the salivary flow. Other
limitations for the use of these agents in the elderly include: unpleasant taste, failure by the
individual to maintain regular use due to lack
of memory, and physical incapacities 10. Incorporation of topical antifungals with tissue conditioner has been shown to inhibit the microbial
plaque formation particularly candidal biofilm
and is effective in prevention of denture stomatitis 11.
By adding silver nanoparticles to GC Soft-Liner tissue conditioner and preparation of disks,
Nam could inhibit colonization of fungi such as
candida (with 0.5 wt% of silver nanoparticles)
and bacteria such as streptococcus mutans and
staphylococcus aureus (with 0.1 wt% of silver
nanoparticles) 12.
Radnai et al also in an investigation by combining the different percentages of Chlorhexidine
and Miconazoles’ gel with Viscogel tissue conditioner, made disks and studied the colonization of Canida albicans, which in their study
only combination of miconazoles’ gel with tissue conditioner was reported effective 13.
With due attention to the usefulness of antifungal combination with tissue conditioner and
also its irrigation by constant washing of saliva
in mouth of denture users, the present study was
aimed to evaluate the maximum effective duration on inhibition of Canida albicans colonization by adding nystatin or fluconazole with
tissue conditioner in an in vitro condition.
Materials and Methods:
A – Disk preparation
In this experimental (Lab-trial) study, the GC
reline tissue conditioner (Soft liner-Japan)
was prepared according to manufacturer’s instruction by addition of Nystatin (5%wt/wt
TC, equivalent to 100,000IU ) or fluconazole
(10%wt/wt ) with tissue conditioner powder 16,
and flattened to 1mm thickness on a sterile surface. Circular thin disks with 5mmm diameter
and 1mm thickness (measured and controlled
by a caliper) were finally prepared using a sterile cutter 14. Similar drug free tissue conditioner
disks were also prepared as control (figure 1).
Disks were then immersed in separate sterile containers containing 100 ml of artificial
saliva, set on a reciprocal shaker (100rpm) at
37°C, and the saliva were regularly changed every day until used for susceptibility testing. In
present study, according to similar studies 15, 16,
and based on the type of study (lab-trial) seven
repetition for each antifungal containing tissue
conditioner disks and duration of incubation
were used.
B - Candida cell suspension preparation
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Stability and duration of antifungal effects of Nystatin and Fluconazole mixed ...
liva were analyzed with SPSS15 software using
statistical ANOVA and Tukey test for multiple
and pairwise comparisons respectively. P value
of less than 0.05 was considered statistically
significant in current study.
Results:
Figure 1-Tissue conditioner disks
To prepare Candida cell suspensions, by cultivation of Canida albicans (ATCC10231) on a
fresh Sabouraud Dextrose agar and incubation
at 30°C for 48 hours, fresh Candida colonies
were prepared. A single colony was used for
preparation of Candida cell suspensions (containing 1×103CFU/ml) in a sterile physiological serum (NaCl 0.85%), using a haemocytometer slide and microscope.
C – Antifungal Susceptibility testing
The agar diffusion test was used for antifungal
susceptibility testing. One hundred μl of Candida cell suspensions was firstly spread onto several Sabouraud dextrose agar plates (streaking
technique). Tissue conditioner disks containing
antifungal and drug free control disks were put
on the surface of plates using sterile forceps by
a sterile forceps 17.
Cultures were incubated at 30°C for 48 hours
and finally the growth inhibitory diameter
around the disks was measured using caliper.
This was repeated 7 times for every tissue conditioner disks containing nystatin, fluconazole
and control drug free disks, for 1, 2, 3 and 5
day intervals.
By repeating the procedures for 7 times for each
of disks containing antifungal drugs in different times, the diameters of inhibitory zones on
Sabouraud dextrose agar were measured using
a caliper and were recorded in tables. The average growth inhibitory zone diameters around
disks in different duration of immersion in sa-
The anti-Candida properties of nystatin or flu
conazole added with the tissue conditioner over
different times were evaluated using the agar
diffusion method (Agar diffusion test). Hereby
tissue conditioner disks containing 100,000 IU
nystatin (with 5% wt of tissue conditioner) as
well as disks containing 10%wt of tissue conditioner with fluconazole 16 were used for evaluation of C. albicans growth and colonization
inhibitory effects.
Antifungal containing tissue conditioner disks
immersed in saliva were inhibited the growth of
C. albicans maximum up to 3 days (equivalent
to 72 hours). However, the inhibitory effect of
nystatin was more in compare with fluconazole
at all studied incubated times.(Table 1)
Table 1- Inhibitory diameters (mm) around tissue
conditioner disks combined with nystatin or fluconazole during different incubation times (N=7)
Time
Combined
antifungal
Diameter of
inhibitory zone
Nystatin
11.2±1.07
24hours
0.0001
Fluconazole
8.6±1.01
Nystatin
2.7±0.7
Fluconazole
1.7±0.56
Nystatin
1.2±0.07
Fluconazole
0.71±0.39
Nystatin
0.15±3815
Fluconazole
0
48hours
72hours
P value
0.023
5days
0.014
0.416
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A.Fallah-tafti, et al.
Table 2- Comparison the average and SD of
the diameters of inhibitory zone, around discs
containing nystatin at paired studied times
Pair wise
comparison
24
48 hours
24
Diameter of
inhibitory zone
P value
2.7±0.7
1.2±0.7
24 hours
11.2±1.07
24
8.6±1.01
48 hours
1.7±0.56
24
8.6±1.01
72 hours
0.7±0.39
24 hours
8.6±1.01
P value
0.0001
0.0001
0.0001
0.0001
0.0001
0.15±0.38
5day
48 hours
0
1.7±0.56
2.7±0.7
0.0001
0.0001
5day
Diameter of
inhibitory zone
11.2±1.07
72 hours
5day
Pair wise
comparison
11.2±1.07
0.0001
48 hours
Table 3- Comparison the average and SD of the
diameters of inhibitory zone, around disks containing fluconazole at paired studied times
0.15±0.38
5day
0
24
1.7±0.56
72 hours
0.7±0.39
72 hours
0.7±0.39
0.006
48
2.7±0.7
hours 72
1.2±0.7
0.001
0.003
5day
72hours
0
1.2±0.7
0.015
5day
0.15±0.38
By comparing the average diameter for inhibitory zone, around disks including nystatin or
fluconazole at different immersing times with
ANOVA statistical test showed only statistically significant differences at 24 (P=0.0001) and
48 hours (P=0.023). But after 72 hours and after 5 days these differences weren’t statistically
significant. (P = 0.1 and 0.4) (Table 1).
By using Tukey statistical test for pairwise
comparisons between the average diameters of
inhibitory zone, around disks combined with
antifungal, it revealed that the statistically significant differences exist between all studied
paired times (Tables 2 and 3).
Discussion:
The efficiency of nystatin or fluconazole incorporated with tissue Conditioner on the growth
of C. albicans over different times was evaluated in present in vitro study. Results showed
that nystatin or fluconazole combined with tissue conditioner in presence of saliva was effective maximmun up to third day and maximum
effect was shown in first 24 hours. In addition,
nystatin showed more topical inhibitory in vitro effect in compare with fluconazole. Several
studies indicated a protective and inhibitory
roles for nyststin or fluconazole added to tissue
conditioner for control and inhibition of Candida colonization, which are in agreement with
the results of present study, although fluconazole responded a slightly weaker than nystatin
9, 10, 16
.
In another study, Falah et al, the inhibitory effect of different weight percentages of nystatin
and fluconazole added with tissue condition-
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Stability and duration of antifungal effects of Nystatin and Fluconazole mixed ...
er, such as 1, 2.5, 5 and 10%wt were studied.
Their results showed that even addition of nystatin equal to one percent of tissue conditioner weight, completely inhibited the growth of
Candida, whereas only addition of fluconazole
equivalent to ten percent of tissue conditioner,
caused complete control of in vitro Candida
colonization 16. Their study also showed the
higher efficiency of nystatin in compare with
fluconazole. However contrary to present study
the role of time wasn’t studied and all results
were evaluated after 48 hours, which are comparable with the results of 48 hours in current
study.
The study of El-Charkawi et al also showed
that the addition of 3, 5, and 10 wt% tissue
conditioner with nystatin was useful to inhibit
the growth and colonization of Candida, and
there is a direct relationship between antifungal weight percentage and their effective time
and the effect of 10 wt% drug is longer than the
rest, and also the weight percent of drug does
not have any effects on tissue conditioner properties18.
Thomas et al in a study compared the effect of
nystatin and amphotricin B in combination with
tissue conditioner, reported nystatin more effective than amphotricin B for inhibition of Candida colonization 19.
In present study, by comparison the inhibitory
effect of nystatin in compare with fluconazole
combined with tissue conditioner at 1, 2, 3 and
5 days, results indicated that each drug containing disks had the ability for controlling of C.
albicans colonization maximum up to 3 days
(equivalent to 72 hours) in presence of artificial saliva. It seems that nystatin or fluconazole
included with tissue conditioner weren’t stable
and reduced by saliva irrigation, to be finished
after three days.
Chow et al in an in vitro study, by combination of 5% wt/wt nystatin, fluconazole and itraconazole with Coe soft tissue conditioner, and
placing on a C. albicans culture, their inhibitory
effect on colonization of Candida were investigated at different times. Maximum growth inhibitory effect of these antifungal was reported
till 3 days. They also reported that their inhibitory effect reach to minimum after 8.2 days 10,
which contradicted with the results of present
study. These contrary results can be as a result
of differences in methods. In Chow study tissue conditioner disks containing antifungal
were directly placed on the surface of cultures
and the culture plates changed every day. But
nystatin and fluconazole contained tissue conditioner disks were immersed in artificial saliva, incubated on rotator shaker (100 rpm) at
37ºC with daily changing of artificial saliva in
current study, comparable with the human oral
cavity condition which can wash out nystatin
and fluconazole faster than tissue conditioner,
and reduce their effects.
Conclusion:
Results of present study indicated that the nystatin or fluconazole incorporated with tissue
conditioner isn’t stable inside the saliva and
their concentration gradually reduced over
time, showed efficient inhibitory effect against
Candida colonization maximmum up to 3 days.
Meanwhile, incorporated nystatin with tissue
conditioner revealed more topical inhibitory
properties than fluconazole in an in vitro condition. Performing an in vivo study using a layer
of tissue conditioner containing nystatin or fluconazole, which fixed under the denture’s mucosal surface in patients with denture stomatitis, over time will suggested. According to the
results of present study, soft liner’s containing
nystatin or fluconazole used under the denture
should be replaced every three days.
Funding/Supports:
This work has been financially supported by
Dentistry school, Shahid Sadoughi University
of Medical Sciences and Health Services as a
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A.Fallah-tafti, et al.
dentistry student thesis (thesis number 425).
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Vol 11,No 1 Spring 2014/25
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