1

UCL SCHOOL OF PHARMACY

BRUNSWICK SQUARE

MSc PFE
Research Project (PHAYGX92)
2017 – 2018

Project title: An investigation to assess the susceptibility of Candida

albicans and Trichophyton mentagrophytes against antifungal agents
for the treatment of Onychomycosis

Student Name: Ruthwik Chegu Ramesh

ID number: 17123081

Supervisor
Supervisor’s name: Dr. Sudaxshina Murdan
Supervisor’s department: Pharmaceutics
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Declaration

The work reported in this report was carried out under the supervision of Dr.
Sudaxshina Murdan in the UCL School of Pharmacy from May to August 2018.
All work is my own unless stated to the contrary and has not been previously
submitted for any degree at UCL or any other university. I have read and
understood UCL’s policies on plagiarism and confirm that I have abided by them
in this report.

Signature: _____________________________

Date: 12th September 2018

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Acknowledgement

Researching and writing this thesis has had an important role in my career at
University College London and I would like to express my gratitude for all the
people without whom I could not have done this.
I would like to thank my supervisor, Dr. Sudaxshina Murdan, who guided me
throughout the process of conducting my research, she always provided me with
the best and most helpful feedback. I would also like to thank my friends and
family who have provided me with the moral and emotional support through my
journey at UCL, never failing to encourage me.
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ABSTRACT

Onychomycosis is a fungal infection of the toe and fingernails, but mostly

common on the toenails. With high prevalence linked to Onychomycosis in
immunocompromised patients and the growing emergence of fungal resistance,
the need for new potent anti-fungal agents is ever so high. Exploring the existing
anti-fungal drugs in combination with one another could be one of the ways to
manufacture drugs better than the existing marketed ones, in terms of better
potency and reduced toxicity. Therefore, this project was mainly aimed at
evaluating the potential of existing anti-fungal drugs and in combination with one
another. Along with this, the anti-fungal activity of silver nanoparticles (Ag-NPs)
were also explored via disk diffusion method. The drugs (Amorolfine HCl,
Terbinafine HCl, Ciclopirox olamine) and nanoparticles were studied against
onychomycosis causing agents, namely Candida albicans (C.albicans) and
Trichophyton mentagrophytes (T.mentagrophytes). Minimum inhibitory
concentration (MIC100, 80 & 50) and fractional inhibitory concentration index (FICI)

were used to assess the combinatory effects of these drugs. The results
highlighted antagonistic effects, between amorolfine HCl and terbinafine HCl
combination, against both C.albicans and T.mentagrophytes.
 5
Table of Contents
ABSTRACT........................................................................................................ 4
1. INTRODUCTION ............................................................................................ 6
2. AIM ................................................................................................................. 9
3. OBJECTIVES................................................................................................. 9
4. MATERIALS AND METHODS ..................................................................... 10
4.1 Fungi species ........................................................................................... 10
4.2 Preparation of stock and test anti-fungal drug solutions ..................... 10
4.3 Preparation of MOPS buffered RPMI-1640 ............................................. 11
4.4 Preparation of SDA petri dishes ............................................................. 11
4.5 Preparation of C.albicans inoculum ....................................................... 11
4.6 Preparation of T.mentagrophytes inoculum .......................................... 12
4.7 Broth microdilution assay for single drug testing ................................ 12
4.8 Broth microdilution assay for combination of drugs testing ............... 13
4.8.1 Preparation of first plate containing drug A: ...................................... 13
4.8.2 Preparation of second plate containing drug B: ................................ 14

4.8.3 Preparation of third plate containing drug A+B.................................. 14

4.9 Influence of Drug combination................................................................ 15
4.10 Disc diffusion test .................................................................................. 16
5. RESULTS AND DISCUSSION ..................................................................... 18
5.1 Evaluation of MIC100 MIC80 and MIC50 of anti-fungal drugs alone and in
combination against C.albicans using the broth microdilution technique 21
5.2 Evaluation of MIC100 MIC80 and MIC50 of anti-fungal drugs alone and in
combination against T.mentagrophytes....................................................... 23
5.3 Comparing the potency of the three anti-fungal drugs between
C.albicans and T.mentagrophytes ................................................................ 26
5.4 Evaluation of the three anti-fungal drugs against T.mentagrophytes
and silver nanoparticles against C.albicans using the disk diffusion
technique ........................................................................................................ 27
5.4.1Potential operational errors committed: .............................................. 29
5.5 Evaluating the efficiency of the two anti-fungal susceptibility testing
methods .......................................................................................................... 32
5.6 Limitations of this study .......................................................................... 32
6. CONCLUSION AND FUTURE WORK ......................................................... 34
REFERENCES:................................................................................................ 35
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1. INTRODUCTION

Onychomycosis is a fungal infection affecting the nails and is responsible for

almost half of all the patients suffering from nail diseases (Gupta, Foley and
Versteeg, 2016) (Gupta, Versteeg and Shear, 2017). This disease is mainly
caused due to dermatophytes T.mentagrophytes and Trichophyton rubrum
(T.rubrum), by mostly causing damages to the toenails. On the contrary, yeasts
such as C.albicans mostly infect the fingernails (Ellis et al., 1997).
Onychomycosis is mostly common among immunocompromised patients, often
develop fungal nail infections at a profound level, therefore requiring them to
resort to lengthy anti-fungal therapy (Mirmirani et al., 2001).

The current choices of treatment for this disease are limited with low cure rates
(Ghannoum and Isham, 2014) and high incidence of recurrence (Gupta and
Simpson, 2012). The existing treatment options are available systematically and
topically. Currently, the most effective oral drug available for this disease is
terbinafine HCl (Tosti and Elston, 2017). But, this oral anti-fungal requires high
doses and long duration of therapy as opposed to topical agents like amorolfine
HCl and ciclopirox olamine (Tabata et al., 2015) (Akhtar, Sharma and Pathak,
2016). Moreover, terbinafine HCl and other oral agents are linked to side effects
such as liver toxicity and drug-drug interactions (Elewski et al., 2005). Therefore,
topical anti-fungal agents are ideal for this disease due to their localised action
and no incidence of drug-drug interactions (Elewski et al., 2005). However, these
are proven to display low efficacy, as a result of the restricted drug permeation
through nails (Gupta, Fleckman and Baran, 2000). The prime barrier to this
limited drug permeation is the compact keratinous cell layers present in the nail
plate (van Hoogdalem et al., 1997). Thus, the current marketed topical
formulations are advised for onychomycosis that are mild and superficial (NICE
CKS, 2018).

Due to antimicrobial resistance on the rise, the need for novel anti-fungal
formulations is ever so high and hence, the investigation into Ag-NPs and
combinational strategies amongst current market drugs is important in achieving
them. In general, discovery and development of new drugs are facing vast
 7
difficulties due to the amount of money and time that is at stake is very high. Thus,
combination therapies and strategies are proving to be productive in elimination
of infections and slowing down the pace of developing drug resistance among
the fungi species (Feng, Xiong and Ran, 2017) (Spitzer, Robbins and Wright,
2016) (Cottarel and Wierzbowski, 2007).

It is well known that combination therapies have proven to be effectual in bacterial

infections like tuberculosis and viral infections like HIV (Ascierto and Marincola,
2011). Similarly, in the case of fungal infections, combination therapies have
proven to exhibit synergistic effects, but only at the in-vitro stage (Evans, 2003).
In specific to onychomycosis, numerous strategies have had their focus on
integrating the topical therapy (for the nail plate) and oral therapy (for the nail
bed) (Evans, 2003). A couple of foregoing studies have demonstrated in-vivo
synergistic effects by combining amorolfine HCl + terbinafine HCl and boasting a
cure rate of 72.3%, as opposed to a cure rate of 37.5% among patients treated
with oral terbinafine HCl only (Olafsson, Sigurgeirrsson and Baran, 2003).
Similarly, another study testing amorolfine HCl, with terbinafine HCl, fluconazole
and itraconazole combinations, showed that 46% of these amorolfine HCl
combinations indicated synergy when tested against yeasts and dermatophytes
(Harman, Ashbee and Evans, 2004). However, certain factors like fungi virulence,
fungi resistance and host immunity, influence the efficacy of a particular drug
combination (Spitzer, Robbins and Wright, 2016). Thus, there is evidence of
synergy in anti-fungal combinations therapy, but unlike anti-bacterial and anti-
viral agents, these evidences and studies are still in their preliminary stages
(Mukherjee et al., 2005).

Parallel to experimenting with anti-fungal drug combinations for treatment of

onychomycosis, silver nanoparticles (Ag-NPs) has attracted some interest in the
quest to treat this disease due to their natural anti-bacterial and anti-inflammatory
properties (Del Rosso, 2014) (Sriramulu and Sumathi, 2017). Ag-NPs are tiny
microscopic particles whose size ranges from 1-100nm (Chen and Schluesener,
2008). They have proven to be effective in humans against certain bacteria and
fungus as they exhibit low toxicity and high potency (in low concerntrations) (Rai,
 8
Yadav and Gade, 2009). Ag-NPs have shown that they act by disrupting the
barrier that stops the entry of drugs i.e, by inhibiting the formation of C.albicans’
biofilm (Lara et al., 2015). The exact mechanism behind the working of Ag-NPs
is quite unclear with different papers suggesting different theories like cell
plasmolysis (Pietrzak et al., 2016), decrease in mycotoxin production in

aspergillus species (Pietrzak et al., 2015), Ag+ disruption of cell membrane (Lara
et al., 2015) etc. As aforementioned, current marketed topical anti-fungal
formulations show poor transungual permeation, but with Ag-NPs this could
potentially be overcome. Transungual delivery is usually influenced by molecular
size and nail permeability decreases as molecular weight increases, indicating

that Ag-NPs and the Ag+ ions they release could exhibit increase in transungual
permeation. Furthermore, a positive ionic charge could result in charge:charge
interactions with keratin (negative charge at physicalogical pH). Therefore, due
to this attraction between charges, it would result in increased drug transport
(Murthy and Maibach, 2013). Nevertheless, this charge:charge interaction
between Ag-NPs and keratin could also decrease the anti-fungal effects, as the
amount of free drug accessible for the fungus to interact with would be reduced
(Zancola, 2017) (Del Rosso, 2014).

The drugs and Ag-NPs were tested against C.albicans and T.mentagrophytes
using broth microdiltuion and disk diffusion methods. In broth microdilution
process, a known quantity of fungi is incubated, in a 96-well plate, with specified
dilutions of anti-fungal agent(s) and ultimately, the minimal inhibitory
concentration (MIC) is obtained. In disk diffusion process, a known quantity of
fungi is incubated, in a petri-dish, with specified dilutions of anti-fungal agents (or
silver nanoparticles) on an anti-microbial disk; the zone of inhibition (ZOI) is
measured using a ruler (Coyle, 2005).
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2. AIM

The aim of this study was to investigate and evaluate susceptibility of C.albicans
and T.mentagrophytes against various anti-fungal agents by broth microdilution
and disk diffusion methods.

3. OBJECTIVES

• To assess the susceptibilities of C.albicans and T.mentagrophytes to

amorolfine HCl, terbinafine HCl and ciclopirox olamine, by calculating and
comparing MIC100,80,50. via broth micro dilution method.

• To investigate whether synergy/additive/antagonistic effect was exhibited while

combining amorolfine HCl, terbinafine HCl and ciclopirox olamine, against
C.albicans and T.mentagrophytes via broth micro dilution method.

• To evaluate the susceptibility of C.albicans against Ag-NPs of various sizes via

disk diffusion.

• To study the susceptibility of T.mentagrophytes against amorolfine HCl,

terbinafine HCl and ciclopirox olamine using the disk diffusion method.
 10

4. MATERIALS AND METHODS

The anti-fungal agents used for this study were amorolfine HCl (Ranbaxy
Research Laboratories, India), terbinafine HCl (AK Scientific, USA) and ciclopirox
olamine (LKT Laboratories, Minnesota, USA). Sabouraud’s dextrose agar (SDA)
(Sigma Aldrich, UK) was used as a medium of growth for all fungi. Roswell Park
Memorial Institute medium-1640 (RPMI-1640) (Sigma Aldrich, UK) was used as
growth medium. The choice of buffer was 3-(N-morpholino)propane sulfonic acid
(MOPS) ((Sigma-Aldrich, UK). Dimethyl sulfoxide (DMSO) (Fisher Scientific, UK)
was used as a solvent; and McFarland 0.5 (Pro-Lab Diagnostics, UK) was used
as a standard during UV spectrophotometry (used to check absorbance of
solutions)

Silver nanoparticles (Ag-NPs) (supplied by Department of Chemical Engineering,

University College London, UK) were used while testing against C.albicans in
disc diffusion experiment.

4.1 Fungi species

The isolates used in this study were C.albicans (ATCC 10231) (Birkbeck,
University of London, UK) and T.mentagrophytes (Birkbeck, University of
London, UK). They were subcultured on SDA petri dishes from existing stocks in
the refrigerator. Prior to the beginning of the experiments, the dishes holding
these fungus were incubated at 320C for 24 hours (C.albicans) and for 7 days
(T.mentagrophytes). These were known as subcultured petri dishes.

4.2 Preparation of stock and test anti-fungal drug solutions

The three anti-fungal agents (powder form) were weighed X100 times the
required amount and dissolved in 1ml of DMSO and these were used as stock
solutions. As the drugs were water insoluble, DMSO was chosen as a solvent.
These stock solutions were diluted 100-fold with MOPS-buffered RPMI-1640, in
order to avoid the effects of DMSO on the fungi species. This resulted in final
 11

concentrations of 64 ㎍/ml for amorolfine HCl, 128 ㎍/ml for terbinafine HCl and

4 ㎍/ml for ciclopirox olamine.

4.3 Preparation of MOPS buffered RPMI-1640

A mass of 10.4g of RPMI-1640 (powder form) was dissolved in 900ml of distilled
water under constant stirring and 34.53g of MOPS buffer was added into this
solution. Upon complete dissolving of these powders, the pH was adjusted to 7.0
using 1mol/L sodium hydroxide. Using distilled water, the volume was made upto
1L. These solutions were autoclaved and eventually stored in refrigerator.

4.4 Preparation of SDA petri dishes

On the weighing balance, 26gm of SDA powder was weighed and suspended in
400ml of water, in a 500ml Duran bottle. The bottles were shaken briefly and
autoclaved at 1210C for 15 minutes. Once they were out of the oven and before
the temperature dropped, the contents were poured into the petri dishes and
allowed to cool.

4.5 Preparation of C.albicans inoculum

Five colonies of microorganisms were picked from the 24-hour old subcultured
petri dishes using a sterile loop and placed into a sterile bijou containing 5ml of
0.85% w/v saline. This mixture was vortexed for 15 seconds, whose absorbance
was then measured with the help of a UV-visible spectrophotometer. Until this
mixture’s turbidity was in compliance with that of 0.5 McFarland standard at
530nm wavelength, more saline or more colonies were added. The spore
suspension was then diluted with RPMI-1640 by a 1000-fold dilution. First, it was
diluted 1:50 to a volume of 1ml and then it was diluted 1:20 to a volume of 20ml
to obtain the final stock suspension. The final colony forming unit (CFU) of the
stock suspension was desired to be at 1x103 - 5x103 CFU/ml.
 12

4.6 Preparation of T.mentagrophytes inoculum

5ml of 0.85% w/v saline was poured into the 7-day old incubated subcultured petri
dish of T.mentagrophytes and the surface was gently scraped with the help of a
sterile loop, in order to release the hyphae and conidia fragments. This mixture
was placed in a sterile bijou and made to stand for 3-5 minutes. The resulting
supernatant liquid containing conidia fragments was transferred to a second
bijou. The contents from the second bijou were filtered using a 5ml syringe,
stainless steel syringe holder and whatman filter paper 40 (diameter 110mm).
The filtrate was vortexed for 15 seconds and its turbidity was measured using a
UV spectrophotometer. More saline was added until the mixture’s turbidity was
in compliance with that of the target absorbance of 0.15-0.17 a.u. This spore
suspension was then diluted 1:50 with RPMI to obtain a concentration of 0.4x104
- 5x104 CFU/ml.

4.7 Broth microdilution assay for single drug testing

In a 96-well U-bottom microtitre plate, 100 ㎕ of RPMI were added in wells from

columns 2-11 with the help of a multichannel pipette and 200 ㎕ of RPMI in

column 12 only (sterility control column). In column 1 from rows A-G, 200 ㎕ of a

test anti-fungal drug solution was added. Column 1 row H (solvent control row)

contained 200 ㎕ of DMSO+RPMI (or 1% DMSO in RPMI) in a 1: 99 dilution and

no drug. With the help of a multichannel pipette, a serial two-fold dilution (double

dilution) was conducted by removing 100 ㎕ from column 1 (from rows A-G) and

transferring them into the following wells of column 2. This process was repeated
until column 10, which would go on to contain the least concentration of the test

drug. 100 ㎕ of the mixture in the wells of column 10 were discarded, therefore

leaving all the wells from columns 1-10 with a quantity of 100 ㎕ of contents only.

This two-fold serial dilution process produced a range of different concentrations

(64-0.25 ㎍/ml for amorolfine HCl, 128-0.5 ㎍/ml for terbinafine HCl and 4-0.125

㎍/ml for ciclopirox olamine) across the 96 well plate, which made a way for
 13

identifying the MIC. This was followed by loading 100 ㎕ of the fungus inoculum

into wells of rows A-H from columns 1-11. Thus, the columns 11 (growth control
column) contained a mixture of fungus inoculum + RPMI only, and not the drug.
Finally, these plates were labelled appropriately, incubated at 320C and visually
examined after 48-hours for C.albicans and 7 days for T.mentagrophytes.

Figure 1: Schematic representation of broth microdilution assay for single drug testing

4.8 Broth microdilution assay for combination of drugs

testing
The preparation of plates for broth microdiltuion assay for combination of drugs
was slightly different and complex, as opposed to the plates prepared for single
drug testing. Three 96-well U-bottom microtitre plates were used in this assay,
first plate to prepare drug A, second plate to prepare drug B and third plate to
combine drugs A+B.

4.8.1 Preparation of first plate containing drug A:

100 ㎕ RPMI was loaded in wells of columns 1-10 from rows B-F and 200 ㎕ of

drug A alone was loaded in only row A of columns 1-10. A two-fold serial dilution
was conducted from rows A-F (down rows i.e., from top to bottom), and finally
 14

100 ㎕ of contents were discarded from wells of row F. This plate was set aside

and the second plate containing drug B was prepared.

4.8.2 Preparation of second plate containing drug B:

100 ㎕ RPMI was loaded in wells of columns 2-9 from rows A-G and 200 ㎕ of

drug B alone was loaded in only wells of column 10 from rows A-G (consisted of
highest concentration of drug, as double dilution was started from here). A two-
fold serial dilution was conducted from wells of column 10 until column 2 (across

columns i.e., from right side to left side), and finally 100 ㎕ of contents were

discarded from wells of column 2. This plate was set aside, and the third plate

was prepared.

4.8.3 Preparation of third plate containing drug A+B

50 ㎕ of drug A was transferred from wells of columns 2-10 between rows A-F,

from the first plate to the exact same wells of third plate. Similarly, 50 ㎕ of drug

B was transferred from wells of columns 2-10 between rows A-F, from the second
plate to the exact same wells of the third plate.

100 ㎕ of drug A was transferred from wells of column 1 from rows A-F, from the

first plate to exact same wells of third plate. Similarly, 100 ㎕ of drug B was

transferred from wells of row G from columns 2-10, from the second plate to the
exact same wells of the third plate. At the end of this, the well in column 1 row G

would contain no drug A or B and was loaded with 100 ㎕ of RPMI.

100 ㎕ of RPMI was loaded in wells of rows A-H from columns 1-11. 100 ㎕ of

RPMI was loaded in wells of column 11 (growth control column) and 200 ㎕ of

RPMI in wells of column 12 (sterility control column). Finally, similar to the single

drug testing plate, 100 ㎕ of the fungus inoculum was added into wells of rows A-

H from columns 1-11. These plates were appropriately labelled, incubated at

 15
320C and visually examined for fungal growth (or lack of growth) after 48-hours
for C.albicans and 7 days for T.mentagrophytes.

Note: At the time of incubation, each of the 96-wells in the microtitre plate would

contain contents of 200 ㎕.

Figure 2: Schematic representation of broth microdilution assay for combination drug testing

After incubation and at the time of visual examination, each well of the 96-well
plate were marked with a score of either 0, 0.5, 1 or 2, depending upon the
amount of growth of C.albicans that was inhibited by the drugs. ‘0’ represented
100% growth inhibition (MIC100), ‘0.5’ represented 80% growth inhibition
(MIC80), ‘1’ represented 50% growth inhibition (MIC50) and ‘2’ represented 0%
growth inhibition by the drugs.
Similar scores were assigned for T.mentagrophytes, but unlike C.albicans, the
emphasis for these plates were on the level of turbidity which were visually
scored.

4.9 Influence of Drug combination

A particular drug combination can produce either of the three effects namely,
synergistic, antagonistic or additive. Synergistic action takes place when the
 16
combined effect of two drugs is greater than the sum of the effect of those drugs
individually. Antagonistic action takes place when the efficacy of two drugs
reduce when combined. Lastly, additive action takes place when the combined
effect of two drugs is equal to the sum of the effect of those drugs individually.
The influence of a drug combination was expressed by using fractional inhibitory
concentration (FIC). This would determine whether a particular drug combination
produced synergistic, antagonistic or additive effect. The sum of both the FIC
values was used to establish the extent of a drug interaction and this was known
as fractional inhibitory concentration index (FICI) (Spitzer, Robbins and Wright,
2016) (Hall, Middleton and Westmacott, 1983). Equations showing the
calculations of FIC and FICI.

FIC A = MIC of drug A in combination with B ⁄MIC of drug A alone

FIC B = MIC of drug B in combination with A ⁄MIC of drug B alone

FICI = FIC A + FIC B

According to the literature, FICI values of ≤ 0.5 indicate synergy, ≥ 4 indicate

antagonism and values between 0.5-4 indicate additive effect (Odds, 2003).

4.10 Disc diffusion test

With the help of a marker, the back of a SDA petri dish was split into 4 quadrants.

Using a sterile spreader, 100 ㎕ of the fungus inoculum (C.albicans or

T.mentagrophytes) was spread throughout the plate. In accordance to the 4

quadrants, three quadrants contained antimicrobial discs loaded with 20 ㎕ of a

particular drug (or Ag-NPs) and the 4th quadrant contained a disc loaded with 20

㎕ of methanol (solvent control). This was followed up with appropriate labelling

and incubation at 320C. The results were checked, after 24 & 48 hours for
C.albicans and 7 days for T.mentagrophytes, by measuring the zone of inhibition
(ZOI) with the help of a ruler.
 17

Figure 3: Schematic representation of disk diffusion testing

 18

5. RESULTS AND DISCUSSION

C.albicans and T.mentagrophytes were tested against three drugs individually

and in their combinations, using the broth micro dilution method. The three drugs
and their combinations included, Amorolfine HCl + Ciclpirox olamine (A + C),
Amorolfine HCl + Terbinafine HCl (A + T) and Terbinafine HCl + Ciclopirox
olamine (T + C). After an incubation period of 48 hours for C.albicans & 7 days
for T.mentagrophytes, their MIC values were obtained. According to the literature,
MIC80 is usually considered for calculating the Geo-mean, FIC and FICI (CLSI,
2017). But, this paper assembles data for MIC100, MIC80 and MIC50, to
enhance understanding and as a learning aid.
 19

Table 1: MIC100, MIC80 and MIC50 values of anti-fungal drugs against C.albicans compared with literature values (Jo Siu et al., 2013) (Li et al., 2004)

C.albicans
Mean MIC Mean MIC Mean MIC
Antifungal drugs 100 /ml Literature MIC 100 80 /ml Literature MIC 80 50 /ml Literature MIC 50
Amorolfine HCL (A) 48.0 not applicable 34.7 <0.125-64 8.8 <0.125-64
Terbinafine HCL (T) 107.3 not applicable 29.3 0.06- >16 9.0 0.06- >16
Ciclopirox olamine (C ) 4.0 not applicable 2.0 0.06-0.5 1.4 0.06-0.5
 20

Table 2: Geometric mean MIC100, MIC80 and MIC50 of anti-fungal drugs, when tested alone and in combination with one another against C.albicans ; along with the

respective percentage change in MIC values post combination testing (Jo Siu et al., 2013) (Li et al., 2004)

C.albicans
G-MIC 100 G-MIC 80 G-MIC 50
G-MIC 100 % change in MIC of G-MIC % change in MIC of G-MIC % change in MIC of
Antifungal drugs /ml combination 80 /ml combination 50 /ml combination
Amorolfine HCL (A) 40.3 not applicable 22.6 not applicable 1.3 not applicable
A in combination with C 0.6 98.51% decrease 0.8 96.46% decrease 0.3 76.92% decrease
A in combination with T 32.0 20.59% decrease 20.2 10.61% decrease 0.5 61.53% decrease

Terbinafine HCL (T) 71.8 not applicable 14.9 not applicable 3.8 not applicable
T in combination with A 3.2 95.54% decrease 3.2 78.52% decrease 2 47.36% decrease
T in combination with C 25.4 64.62% decrease 40.3 170.47% increase 1.6 57.89% decrease

Ciclopirox olamine (C ) 4.0 not applicable 1.2 not applicable 0.6 not applicable
C in combination with A 3.2 20% decrease 0.2 83.33 decrease 0.2 66.66% decrease
C in combination with T 1.3 67.5% decrease 0.2 83.33 decrease 0.1 83.33% decrease
 21

5.1 Evaluation of MIC100 MIC80 and MIC50 of anti-fungal

drugs alone and in combination against C.albicans using
the broth microdilution technique
Ciclopirox olamine (4 ㎍/ml) seemed to be the most potent drug, followed by

amorolfine HCl (40.3 ㎍/ml) and then terbinafine HCl (71.8 ㎍/ml) (table 1 & 2).

These values seem to be higher than usual for drugs being tested against
C.albicans, but it cannot be conclusive since the data related to MIC100 was not
collected or compiled often by other research studies and literatures, therefore it
could not be compared to any existing values. But, it is interesting to note that
ciclopirox olamine is more potent than amorolfine HCl and terbinafine HCl. A
potential reason for this could be that ciclopirox olamine has different modes of
action and drug targets on fungi’s cells, as compared to amorolfine and
terbinafine HCl. Ciclopirox olamine targets enzymes that are metal dependent,
which are necessary for production of energy and mitochondrial electron
transport processes in fungi cells. This is very different compared to amorolfine
and terbinafine HCl, which target sterol synthesis in fungi membranes. Also,
ciclopirox olamine exercises its effect by disturbing DNA repair and cell division
signals. Studies in the past have demonstrated excellent efficacy of ciclopirox
olamine, as compared to the allylamines and azoles (Gupta, Fleckman and
Baran, 2000) (Paul et al., 2013). The obtained data (table 1 & 2) falls within the
literature data range (Jo Siu et al., 2013) (Li et al., 2004), except with respect to
ciclopirox olamine whose MIC80 and MIC50 values are 40x and 28x respectively,
more than the literature values. The reason for this could be a number of factors
like operational errors during the experiment, different strains of C.albicans used
or differences in the CFU/ml used, as opposed to the ones used in the literatures
etc. Similar to the findings from MIC100, there seems to be consistency in results

of MIC80 and MIC50 with ciclopirox olamine (1.2 ㎍/ml) being the most potent

drug acting against C.albicans followed by terbinafine HCl (14.9 ㎍/ml) and

amorolfine HCl (22.6 ㎍/ml) with the lowest potency amongst the three drugs. As

aforementioned, by targeting such crucial cellular processes, it explains as to why

 22
ciclopirox olamine’s MIC is the lowest and its capability in destroying C.albicans
is more effective than other two drugs.
Mostly, there was a certain percentage of decrease in MIC100 MIC80 and MIC50
when the drugs were combined with one another, as seen in table 2. This dip in
percentage of MICs indicated greater efficiency of drugs when in combination
than when used individually.

Table 3: FICI analysis at MIC100, MIC80 and MIC50 of anti-fungal drugs combinations against
C.albicans

C.albicans
Antifungal drug combination FICI Result
A+C 1.1 additive
MIC 100 A+T 1.3 synergism
C+T 1.0 additive
A+C 0.6 additive
MIC 80 A+T 4.5 antagonism
C+T 3.2 additve
A+C 1.1 additive
MIC 50 A+T 1.1 additive
C+T 0.4 synergism

At MIC100, MIC80 and MIC50, an additive effect amongst A+C combination with
FICI values of 0.9, 0.6 and 1.1 (table 3) respectively, seemed to be consistent.
However, the combinatory effects between A+T and C+T at MIC100, MIC80 and
MIC50 seemed to be inconsistent, with all three effects of synergism, additive
and antagonism observed. The reason for the same drugs in combination tested
against the same fungi species in the same micro-titre plate, yet producing polar
opposite combinatory effects in MIC100, MIC80 and MIC50 is still unclear and
would require further studies. The antagonistic effect observed in MIC80 A+T
combination, with a FICI value of 4.5 (table 3), could be due to the interfering
mechanisms of actions of the two anti-fungals, where in the action of one drug
masks the effect of other drug (antagonistic buffering) or one drug completely
suppresses the action of the other drug (antagonistic suppression) (Yin et al.,
2014). As previously seen with β-lactum combinations, the antagonistic effect
could be due to induction of resistant genes (Acar, 2000). Furthermore, previous
studies have shown that by combining bacteriostatic and bactericidal drugs, an
 23
antagonistic effect was produced (Ocampo et al., 2014). This could be applicable
to anti-fungal agents as well, as fungistatic agents inhibit fungus growth, however
fungicidal agents need growing fungus to exhibit their mechanism of action. On
the contrary, Chait et al showed some beneficial effects of antagonistic anti-biotic
combinations in the management and delay of resistant effects, as a
consequence of competitive selection against resistant alleles (Chait, Craney and
Kishony, 2007). Therefore, it is important to note this and explore these beneficial
effects of antagonism in further studies of these antifungal combinations

5.2 Evaluation of MIC100 MIC80 and MIC50 of anti-fungal

drugs alone and in combination against T.mentagrophytes
Terbinafine HCl (0.6 ㎍/ml/ml) seemed to be the most potent drug, followed by

amorolfine HCl and ciclopirox olamine at 4 ㎍/ml (table 4 & 5). Similar to MIC100

data of C.albicans, these data too cannot be conclusive, as MIC100 data was not
obtained by earlier studies; hence it could not be compared to any pre-existing
values. However, MIC80 data (table 4 & 5) achieved for the three anti-fungal
drugs agrees with the literature values (Adimi et al., 2013) and similar to MIC100,

terbinafine HCl(0.8 ㎍/ml) appeared to be the most potent drug acting against

T.mentagrophytes followed by amorolfine HCl (2.5 ㎍/ml) and then ciclopirox

olamine(4.0 ㎍/ml). With the literatures suggesting terbinafine HCl to be one of

the most effective agent for onychomycosis in elders (Dogra, Kaul and Yadav,
2017), this study seems to be dependable. Also, the order of potency of the above
drugs against T.mentagrophytes seems to be consistent with studies conducted
by (Wiederhold et al., 2014) and (Adimi et al., 2013), once again indicating the
reliability of this study. However, it is interesting to note that the values obtained
in this study seem to be slightly higher compared to the ones listed in the
literature; some possible reasons for this could be due to anti-fungal susceptibility
differing amongst populations or use of different dermatophyte strains (Tamura
et al., 2014).
 24

Table 4: MIC100, MIC80 and MIC50 values of anti-fungal drugs against T.mentagrophytes compared with literature values (Gupta et al.,2003) ((Jo Siu et al., 2013)

(Li et al., 2004) (Koga et al., 2009) (Adimi et al., 2013)

T.mentagrophytes
Mean MIC Mean MIC Mean MIC
Antifungal drugs 100 /ml Literature MIC 100 80 /ml Literature MIC 80 50 /ml Literature MIC 50
0.004-0.06
0.031-0.25
Amorolfine HCL (A) 7.8 not applicable 6.8 0.0078-32 6.5 0.02- >0.08
0.003-0.5
0.004-0.5
Terbinafine HCL (T) 0.7 not applicable 1.8 0.0156-16 5.6 0.002-0.031
0.03-0.125
0.03-0.5
Ciclopirox olamine (C ) 4.0 not applicable 4.0 0.0312-32 4.0
 25

Table 5: Geometric mean MIC100, MIC80 and MIC50 of anti-fungal drugs, when tested alone and in combination with one another against T.mentagrophytes ; along

with the respective percentage change in MIC values post combination testing Gupta et al.,2003) ((Jo Siu et al., 2013) (Li et al., 2004) (Koga et al., 2009) (Adimi et
al., 2013)

T.mentagrophytes
G-MIC 100 G-MIC 80 G-MIC 50
G-MIC 100 % change in MIC of G-MIC % change in MIC of G-MIC % change in MIC of
Antifungal drugs /ml combination 80 /ml combination 50 /ml combination
Amorolfine HCL (A) 4.0 not applicable 2.5 not applicable 1.8 not applicable
A in combination with C 4.0 0% change 2.5 0% change 0.8 55.55% decrease
A in combination with T 0.5 87.5% decrease 0.8 68% decrease 0.8 55.55% decrease

Terbinafine HCL (T) 0.6 not applicable 0.8 not applicable 2.2 not applicable
T in combination with A 2 233.33% increase 6.3 687.5% increase 6.3 186.36% increase
T in combination with C 0.5 16.66% decrease 1 25% increase 2 9.09% decrease

Ciclopirox olamine (C ) 4.0 not applicable 4.0 not applicable 4.0 not applicable
C in combination with A 0.1 97.5% decrease 0.4 90% decrease 1 75% decrease
C in combination with T 0.1 97.5 decrease 0.3 92.5% decrease 0.8 80% decrease
 26
At MIC100, MIC80 and MIC50, the additive effect amongst A+C combination with
FICI values of 1.1, 1,4 and 1.2 respectively (table 6), seems to be consistent.
MIC80 and MIC50 A+T combinations exhibited an antagonistic effect. In contrast
to this result, a previous study proved and displayed synergy between A+T
combination in 29% of dermatophyte isolates, which included T.mentagrophytes
(Harman, Ashbee and Evans, 2004). As aforementioned, the resulted
antagonistic effects from this study could potentially be owed to the fact that two
drugs’ mechanisms of actions interfering with one another (Yin et al., 2014). Also,
it is interesting to note from table 5, that when terbinafine HCl was combined with
amorolfine HCl, an increase of 233.33% 687.5% and 180.36% was seen at
MIC100, MIC80 and MIC50 respectively. This meant that the efficiency of
terbinafine HCl decreased while in combination with amorolfine HCl, supporting
the earlier studies made by Yin et al. But further research and repeated trials of
this combination could rationalize the antagonistic effect observed between the
A+T.

Table 6: FICI analysis at MIC100, MIC80 and MIC50 of anti-fungal drugs combinations against
T.mentagrophytes

T.mentagrophytes
Antifungal drug combination FICI Result
A+C 1.1 additive
A+T 1.3 additive
MIC 100 C+T 1.0 additive
A+C 1.4 additive
A+T 13.3 antagonism
MIC 80 C+T 0.9 additve
A+C 1.2 additive
A+T 13.0 antagonism
MIC 50 C+T 3.3 additve

5.3 Comparing the potency of the three anti-fungal drugs

between C.albicans and T.mentagrophytes
It was noted that the mean MIC80 values of the three drugs acting against
T.mentagrophytes seemed to be lower than those compared to the values
against C.albicans. This meant that the three drugs potentially portrayed higher
potency against T.mentagrophytes as compared to C.albicans. This fact
 27
appeared to be in agreement with previous in-vitro studies and literatures (Bueno
et al., 2009) (Jo Siu et al., 2013). However, this differed from previous clinical
experience, wherein amorolfine HCl was noted to be exhibit low effectivity against
dermatophytes as compared to yeasts (NICE CKS, 2018). Besides, in particularly
with respect to terbinafine HCl, it seemed to show a higher potency against
T.mentagrophytes as compared to C.albicans. This was anticipated, as it is
generally recommended as the first-line of treatment in dermatophyte nail
infections and as the secondary alternate in treatment of Candida infections of
nails (NICE CKS, 2018).
Moreover, all agents showed lower MIC80 against T.mentagrophytes as
compared to C.albicans, except for ciclopirox olamine. These differences in
susceptibility observed in dermatophytes and non-dermatophytes highlights the
need to identify the causative fungi species in order to treat onychomycosis. This
is essential in order to enable the right selection of effectual anti-fungal agents,
therefore decreasing the chances of developing anti-microbial resistance
(Tamura et al., 2014).

5.4 Evaluation of the three anti-fungal drugs against

T.mentagrophytes and silver nanoparticles against
C.albicans using the disk diffusion technique
The incubated petri dishes of T.mentagrophytes were tested against Amorolfine
HCl, Terbinafine HCl and Ciclopirox olamine; and C.albicans were tested against
silver nanoparticles. These dishes were removed and checked for zone of
inhibitions. The tables (7 & 8) display the average zone of inhibition (ZOI)
amongst the three disks for a particular drug in a single plate. In the case of
T.mentagrophytes, the results of all three anti-fungal drugs are not consistent with
the literature values (Rizi et al., 2015). This variation in results from previous
studies could be attributed to the type of medium used, incubation temperature,
incubation period, pH, inoculum size and end point criteria (Singh et al., 2007).
Factors such as composition of culture media, utilization of nutritional
supplements and agar diffusion of drugs could be potential reasons for the poor
inhibitory activity observed in ciclopirox olamine. The medium’s composition
 28
might have had a crucial role in the poor activity of ciclopirox olamine, as its

mechanism of action depends on chelation of ions like Fe2+ (Singh et al., 2007).
Some insignificant values were obtained due to complete (full) ZOI. There were
several zero ZOIs observed in both cases, drugs and silver nanoparticles (table
7 & 8). In principle, zero ZOI means that the fungus has colonized the area and
its growth was not inhibited by that particular drug.
As figure (4 & 5) shows, the silver nanoparticles exhibited no inhibition of growth
of the C.albicans. A probable reason for this could be that the concentration of
Ag-NPs was too low to cause any kill. It is also possible that the size of Ag-NPs
was too large to exhibit any anti-fungal effect when tested in SDA, as they were
probably unable to diffuse through the agar medium. This was supported by
previous studies conducted by Agnihotri et al (Agnihotri, Mukherji and Mukherji,
2014).
Table 7: Average ZOI (cm) for various anti-fungal drugs tested against T.mentag using disk
diffusion method (Rizi et al., 2015)

T.mentag
Average ZOI Literature
Antifungal drugs (cm) values
Amorolfine HCL 0 9.00 ± 0.00
Terbinafine HCL 0 7.95 ± 0.32
Ciclopirox olamine 1 not applicable
Ciclopirox olamine at 50 /disk 1.66 5.39 ± 0.31

Table 8: Average ZOI (cm) after 24 & 48 hours for Ag-NPs of different sizes tested against
C.albicans using disk diffusion method

C.albicans
Antifungal Average ZOI after 24 hours Average ZOI after 48 hrs
drugs (cm) (cm)
RB429ii 0 0
RB434ii 0 0
RB433iii 0 0
RB433i 0 0
RB431i 0 0
RB433ii 0 0
RB430i 0 0
RB430iii 0 0
RB430ii 0 0
RB429iii 0 0
RB429i 0 0
 29

5.4.1Potential operational errors committed:

a) Disks:
In order to minimise the possibilities of condensation and drug concentration
getting diluted in the disks, generally they are supposed to be equilibrated at room
temperature for at least 15 minutes or more, before being incubated (Buller,
Thomas and Barton, 2014). This was not followed during the experiment and
could potentially be a reason for dishes exhibiting no ZOI.

Figure 4: Ag-NPs discs against C.albicans petri dish after 24 hours of incubation, exhibiting no
ZOI
 30
Figure 5: Ag-NPs discs against C.albicans petri dish after 48 hours of incubation, still exhibiting
no ZOI

b) Inoculum growth:
The cell density is considered to be ideal when the colonies touch each other,
and cell density is considered to be too light (non-ideal) when the colonies are
isolated. Incorrect inoculum concentration causes the growth of fungus (post-
incubation) to be too thick or too thin, which directly affects the accuracy of zone
sizes or no zone at all (Buller, Thomas and Barton, 2014).
 31
Figure 6 (above): Terbinafine HCl discs against T.mentagrophytes petri dish after 7 days of
incubation, exhibiting isolated colonies and ZOI > 8.9cm

Figure 7 (below): Ciclopirox olamine (50 ㎍/disk) discs against T.mentagrophytes petri dish after

7 days of incubation, exhibiting non-spherical zone shape and size

Figure 8 (below): Ciclopirox olamine (5 ㎍/disk) discs against T.mentagrophytes petri dish after 7

days of incubation, exhibiting thick fungus growth on top of the disc

 32
c) Time constraints:
The above listed operational errors were easily identified and could have been
corrected. However, due to lack of time, the experiments were not able to be
repeated for rectification, as in certain cases like T.mentagrophytes, the minimum
incubation time taken is 7 days.

5.5 Evaluating the efficiency of the two anti-fungal

susceptibility testing methods
Unlike the inconsistent fungi growth during the disk diffusion experiments, there
was consistent optimal growth of fungi seen throughout broth microdilution
process. A potential reason for this could be due to the different mediums used
between the two. The RPMI used in microdilution process, organisms are able to
source desired nutrients from them and grow efficiently (Weerasekera et al.,
2016). However, in disk diffusion, the organisms being embedded to the SDA
could have possibly caused deprivation in oxygen levels and eventually limiting
the fungi growth (Weerasekera et al., 2016). Therefore, affecting the final results.
The values (MICs and G-MICs) obtained from broth microdilution seemed to be
consistent with the literature data, unlike the results of disk diffusion, wherein a
single value was not consistent with those compared to the literatures.
This study suggests the superiority of broth microlution process over disk
diffusion, based on the mediums (RPMI-1640 and SDA respectively) used for the
growth of fungus.

5.6 Limitations of this study

• Throughout the study, only a single strain of C.albicans or T.mentagrophytes
was tested. This could have impacted the results, as they are not true
representation of the overall population of the organisms.

• The MIC and G-MIC values determined were compared with other literature
values that used different fungal strains as opposed to the ones used in this
study.

• In broth microdilution and disc diffusion, occasionally the fungal growth wasn’t
clearly visible which could have possibly resulted in inconsistent data
 33
interpretation and final MIC results. Novel methods like SPOTi assay could be
used to overcome this concern, and the results of both could be compared and
contrasted. SPOTi assays provide more reliable MIC data, as the drug is mixed
thoroughly in agar and this provides a uniform exposure of drug concentrations
towards the fungi colonies (Rizi et al., 2015).

• Reading of 96-well plates were done visually as opposed to using a mirror

reader or spectrophotometer (Pharma, 2018). This could have potentially
caused few discrepancies in results due to operator subjectivity.

• In disk diffusion experiments, this study employed SDA as the medium for
growth of fungus. SDA is known to adequately support growth of different
yeasts and dermatophytes, however, their ability to produce clear inhibition
zone edges has not been evaluated. The recommended medium in CLSI
reference assay is Mueller-Hinton (MH) agar with 2% glucose and methylene
blue. MH agar medium has been evaluated to produce clear inhibition zone
edges and less intrazonal growth (Peano et al., 2017).
 34

6. CONCLUSION AND FUTURE WORK

Combination drug strategies were investigated to better the cure rates for
onychomycosis, decrease the duration of therapies and delay the emergence of
resistance. The study study showed ciclopirox olamine to be the most potent drug
against C.albicans and terbinafine HCl to be the most potent drug against
T.mentagrophytes. There was no proof of consistent synergistic effects between
the drugs amorolfine HCl, terbinafine HCl and ciclopirox olamine against
C.albicans and T.mentagrophytes. Effects of antagonism, against both
C.albicans and T.mentagrophytes, was observed between amorolfine HCl and
terbinafine HCl combinations. Also, the three drugs tested seemed to show
greater potency against T.mentagrophytes, as compared to C.albicans. The
results of disk diffusion were noted to be inconsistent and the method proved to
be inferior to the broth microdilution process. However, the barriers associated
with nail permeation will require further studies and research, in order to improve
the efficiency of drugs and them in combinations. Also, the differences in
susceptibility observed in dermatophytes and non-dermatophytes highlights the
need to research and identify the causative fungi species in order to treat
onychomycosis. This is essential in order to enable the right selection of effectual
anti-fungal agents, therefore decreasing the chances of developing anti-microbial
resistance.
 35
REFERENCES:

Acar, J. (2000). ANTIBIOTIC SYNERGY AND ANTAGONISM. Medical Clinics

of North America, 84(6), pp.1391-1406.

Adimi, P., Hashemi, S., Mahmoudi, M., Mirhendi, H., Shidfar, M., Emmami, M.,
Rezaei-Matehkolaei, A., Gramishoar, M. and Kordbacheha, P. (2013). In-vitro
Activity of 10 Antifungal Agents against 320 Dermatophyte Strains Using
Microdilution Method in Tehran. Iran J Pharm.

Agnihotri, S., Mukherji, S. and Mukherji, S. (2014). Size-controlled silver

nanoparticles synthesized over the range 5–100 nm using the same protocol
and their antibacterial efficacy. RSC Adv., 4(8), pp.3974-3983.

Akhtar, N., Sharma, H. and Pathak, K. (2016). Onychomycosis: Potential of Nail

Lacquers in Transungual Delivery of Antifungals. Scientifica, 2016, pp.1-12.

Ascierto, P. and Marincola, F. (2011). Combination therapy: the next opportunity

and challenge of medicine. Journal of Translational Medicine, 9(1), p.115.

Bueno, J., Martinez, C., Zapata, B., Sanclemente, G., Gallego, M. and Mesa, A.
(2009). In vitro activity of fluconazole, itraconazole, voriconazole and terbinafine
against fungi causing onychomycosis. Clinical and Experimental Dermatology,
35(6), pp.658-663.

Buller, N., Thomas, A. and Barton, M. (2014). Antimicrobial Susceptibility

Testing. [online] Agriculture.gov.au. Available at:
http://www.agriculture.gov.au/SiteCollectionDocuments/animal/ahl/ANZSDP-
Antimicrobial-susceptibility-testing.pdf [Accessed 30 Aug. 2018].

Chait, R., Craney, A. and Kishony, R. (2007). Antibiotic interactions that select
against resistance. Nature, 446(7136), pp.668-671.
 36
Chen, X. and Schluesener, H. (2008). Nanosilver: A nanoproduct in medical
application. Toxicology Letters, 176(1), pp.1-12.

CLSI (2017). [online] Available at:

https://clsi.org/media/1897/m27ed4_sample.pdf [Accessed 30 Aug. 2018].

Cottarel, G. and Wierzbowski, J. (2007). Combination drugs, an emerging

option for antibacterial therapy. Trends in Biotechnology, 25(12), pp.547-555.

Coyle, M. (2005). Manual of Antimicrobial Susceptibility Testing. [online]

Asm.org. Available at:
https://www.asm.org/ccLibraryFiles/FILENAME/000000002484/Manual%20of%
20Antimicrobial%20Susceptibility%20Testing.pdf [Accessed 5 Sep. 2018].

Del Rosso, J. (2014). The Role of Topical Antifungal Therapy for

Onychomycosis and the Emergence of Newer Agents. Journal of clinical and
aesthetic dermatology. [online] Available at:
https://www.ncbi.nlm.nih.gov/pubmed/25053979 [Accessed 30 Aug. 2018].

Elewski, B.E., & Tavakkol, A. (2005). Safety and tolerability of oral antifungal
agents in the treatment of fungal nail disease: a proven reality. Therapeutics
and Clinical Risk Management, 1, 299 - 306.

Ellis, D., Watson, A., Marley, J. and Williams, T. (1997). Non-dermatophytes in

onychomycosis of the toenails. British Journal of Dermatology, 136(4), pp.490-
493.

Evans, E. (2003). Drug synergies and the potential for combination therapy in
onychomycosis. British Journal of Dermatology, 149(s65), pp.11-13.

Feng, X., Xiong, X. and Ran, Y. (2017). Efficacy and tolerability of amorolfine
5% nail lacquer in combination with systemic antifungal agents for
 37
onychomycosis: A meta-analysis and systematic review. Dermatologic Therapy,
30(3), p.e12457.

Ghannoum, M. and Isham, N. (2014). Fungal Nail Infections (Onychomycosis):

A Never-Ending Story?. PLoS Pathogens, 10(6), p.e1004105.

Gupta, A. and Kohli, Y. (2003). In vitro susceptibility testing of ciclopirox,

terbinafine, ketoconazole and itraconazole against dermatophytes and
nondermatophytes, and in vitro evaluation of combination antifungal
activity. British Journal of Dermatology, 149(2), pp.296-305.

Gupta, A. and Simpson, F. (2012). New therapeutic options for

onychomycosis. Expert Opinion on Pharmacotherapy, 13(8), pp.1131-1142.

Gupta, A., Fleckman, P. and Baran, R. (2000). Ciclopirox nail lacquer topical
solution 8% in the treatment of toenail onychomycosis. Journal of the American
Academy of Dermatology, 43(4), pp.S70-S80.

Gupta, A., Foley, K. and Versteeg, S. (2016). New Antifungal Agents and New
Formulations Against Dermatophytes. Mycopathologia, 182(1-2), pp.127-141.

Gupta, A., Versteeg, S. and Shear, N. (2017). Onychomycosis in the 21st

Century: An Update on Diagnosis, Epidemiology, and Treatment. Journal of
Cutaneous Medicine and Surgery, 21(6), pp.525-539.

Hall, M., Middleton, R. and Westmacott, D. (1983). The fractional inhibitory

concentration (FIC) index as a measure of synergy. Journal of Antimicrobial
Chemotherapy, 11(5), pp.427-433.

Harman, S., Ashbee, H. and Evans, E. (2004). Testing of antifungal

combinations against yeasts and dermatophytes. Journal of Dermatological
Treatment, 15(2), pp.104-107.
 38
Jo Siu, W., Tatsumi, Y., Senda, H., Pillai, R., Nakamura, T., Sone, D. and
Fothergill, A. (2013). Comparison ofIn VitroAntifungal Activities of Efinaconazole
and Currently Available Antifungal Agents against a Variety of Pathogenic Fungi
Associated with Onychomycosis. Antimicrobial Agents and Chemotherapy,
57(4), pp.1610-1616.

Koga, H., Nanjoh, Y., Makimura, K. and Tsuboi, R. (2009). In vitroantifungal

activities of luliconazole, a new topical imidazole. Medical Mycology, 47(6),
pp.640-647.

Lara, H., Romero-Urbina, D., Pierce, C., Lopez-Ribot, J., Arellano-Jiménez, M.

and Jose-Yacaman, M. (2015). Effect of silver nanoparticles on Candida
albicans biofilms: an ultrastructural study. Journal of Nanobiotechnology, 13(1).
Leem, S., Park, J., Kim, I., Chae, J., Sugino, A. and Sunwoo, Y. (2003). The
possible mechanism of action of ciclopirox olamine in the yeast Saccharomyces
cerevisiae. Mol. Cells, pp.55-61.

Li, R., Wan, Z., Wang, A., Shen, Y., Lu, C., Li, M., Xi, L., Liu, W. and Zeng, F.
(2004). In vitro susceptibility testing of amorolfine in pathogenic fungi isolated
from dermatomycosis patients in China. In vitro Empfindlichkeitsprufung mit
Amorolfin an pathogenen Pilzen von Patienten mit Dermatomykosen in
China. Mycoses, 47(9-10), pp.402-406.

Mirmirani, P., Hessol, N., Maurer, T., Berger, T., Nguyen, P., Khalsa, A.,
Gurtman, A., Micci, S., Young, M., Holman, S., Gange, S. and Greenblatt, R.
(2001). Prevalence and predictors of skin disease in the Women's Interagency
HIV Study (WIHS). Journal of the American Academy of Dermatology, 44(5),
pp.785-788.

Mukherjee, P., Sheehan, D., Hitchcock, C. and Ghannoum, M. (2005).

Combination Treatment of Invasive Fungal Infections. Clinical Microbiology
Reviews, 18(1), pp.163-194.
 39
Murthy, S. and Maibach, H. (2013). Topical nail products and ungual drug
delivery. 1st ed. Boca Raton: CRC Press.

NICE CKS (2018). Fungal nail infection - NICE CKS. [online] Available at:
https://cks.nice.org.uk/fungal-nail-infection [Accessed 30 Aug. 2018].

Ocampo, P., Lázár, V., Papp, B., Arnoldini, M., Abel zur Wiesch, P., Busa-
Fekete, R., Fekete, G., Pál, C., Ackermann, M. and Bonhoeffer, S. (2014).
Antagonism between Bacteriostatic and Bactericidal Antibiotics Is
Prevalent. Antimicrobial Agents and Chemotherapy, 58(8), pp.4573-4582.

Odds, F. (2003). Synergy, antagonism, and what the chequerboard puts

between them. Journal of Antimicrobial Chemotherapy, 52(1), pp.1-1.

Olafsson, J., Sigurgeirrsson, B. and Baran, R. (2003). Combination therapy for

onychomycosis. British Journal of Dermatology, [online] pp.15-18. Available at:
https://www.ncbi.nlm.nih.gov/pubmed/14510971 [Accessed 30 Aug. 2018].

Paul, C., Coustou, D., Lahfa, M., Bulai-Livideanu, C., Doss, N., Mokthar, I.,
Turki, H., Nouira, R., Fazaa, B., Ben Osman, A., Zourabichvili, O., Cazeau, C.,
Coubetergues, H., Picot, S., Bienvenu, A. and Voisard, J. (2013). A Multicenter,
Randomized, Open-Label, Controlled Study Comparing the Efficacy, Safety and
Cost-Effectiveness of a Sequential Therapy with RV4104A Ointment,
Ciclopiroxolamine Cream and Ciclopirox Film-Forming Solution with Amorolfine
Nail Lacquer Alone in Dermatophytic Onychomycosis. Dermatology.

Peano, A., Pasquetti, M., Tizzani, P., Chiavassa, E., Guillot, J. and Johnson, E.
(2017). Methodological Issues in Antifungal Susceptibility Testing of Malassezia
pachydermatis. Journal of Fungi, 3(3), p.37.

Pietrzak, K., Glińska, S., Gapińska, M., Ruman, T., Nowak, A., Aydin, E. and
Gutarowska, B. (2016). Silver nanoparticles: a mechanism of action on
moulds. Metallomics, 8(12), pp.1294-1302.
 40

Pietrzak, K., Twarużek, M., Czyżowska, A., Kosicki, R. and Gutarowska, B.

(2015). Influence of silver nanoparticles on metabolism and toxicity of
moulds. Acta Biochimica Polonica, 62(4), pp.851-857.

Polak, A. (1992). Preclinical Data and Mode of Action of

Amorolfine. Dermatology, 184(1), pp.3-7.

Rai, M., Yadav, A. and Gade, A. (2009). Silver nanoparticles as a new

generation of antimicrobials. Biotechnology Advances, 27(1), pp.76-83.

RYDER, N. (1992). Terbinafine: Mode of action and properties of the squalene

epoxidase inhibition. British Journal of Dermatology, 126(s39), pp.2-7.

Singh, J., Singh, J., Zaman, M. and Gupta, A. (2007). Evaluation of

microdilution and disk diffusion methods for antifungal susceptibility testing of
dermatophytes. Medical Mycology, 45(7), pp.595-602.

Spitzer, M., Robbins, N. and Wright, G. (2016). Combinatorial strategies for

combating invasive fungal infections. Virulence, 8(2), pp.169-185.

Spitzer, M., Robbins, N. and Wright, G. (2016). Combinatorial strategies for

combating invasive fungal infections. Virulence, 8(2), pp.169-185.

Sriramulu, M. and Sumathi, S. (2017). Photocatalytic, antioxidant, antibacterial

and anti-inflammatory activity of silver nanoparticles synthesised using forest
and edible mushroom. Advances in Natural Sciences: Nanoscience and
Nanotechnology, 8(4), p.045012.

Tabata, Y., Takei-Masuda, N., Kubota, N., Takahata, S., Ohyama, M., Kaneda,
K., Iida, M. and Maebashi, K. (2015). Characterization of Antifungal Activity and
Nail Penetration of ME1111, a New Antifungal Agent for Topical Treatment of
Onychomycosis. Antimicrobial Agents and Chemotherapy, 60(2), pp.1035-1039.
 41

Tamura, T., Asahara, M., Yamamoto, M., Yamaura, M., Matsumura, M., Goto,
K., Rezaei-Matehkolaei, A., Mirhendi, H., Makimura, M. and Makimura, K.
(2014). In vitrosusceptibility of dermatomycoses agents to six antifungal drugs
and evaluation by fractional inhibitory concentration index of combined effects
of amorolfine and itraconazole in dermatophytes. Microbiology and
Immunology, 58(1), pp.1-8.

Tosti, A. and Elston, D. (2017). Onychomycosis Guidelines: Guidelines

Summary. [online] Emedicine.medscape.com. Available at:
https://emedicine.medscape.com/article/1105828-guidelines [Accessed 30 Aug.
2018].

van Hoogdalem, E., van den Hoven, W., Terpstra, I., van Zijtveld, J., Verschoor,
J. and Visser, J. (1997). Nail penetration of the antifungal agent oxiconazole
after repeated topical application in healthy volunteers, and the effect of
acetylcysteine. European Journal of Pharmaceutical Sciences, 5(3), pp.119-
127.

Weerasekera, M., Wijesinghe, G., Jayarathna, T., Gunasekara, C., Fernando,

N., Kottegoda, N. and Samaranayake, L. (2016). Culture media profoundly
affect Candida albicans and Candida tropicalis growth, adhesion and biofilm
development. Memórias do Instituto Oswaldo Cruz, 111(11), pp.697-702.

Wiederhold, N., Fothergill, A., McCarthy, D. and Tavakkol, A. (2014).

Luliconazole Demonstrates PotentIn VitroActivity against Dermatophytes
Recovered from Patients with Onychomycosis. Antimicrobial Agents and
Chemotherapy, 58(6), pp.3553-3555.

Yin, N., Ma, W., Pei, J., Ouyang, Q., Tang, C. and Lai, L. (2014). Synergistic
and Antagonistic Drug Combinations Depend on Network Topology. PLoS
ONE, 9(4), p.e93960.
 42
Zancola, S. (2017). New topical formulation for onychomycosis:
transfersomes containing terbinafine HCL. Thesis (masters), University of
Trieste.
 43
APPENDIX 1:

Single Drug Test

Studen Date Dru Fungi Experimen MIC 100 MIC 80 MIC 50
t ID plate read g species t /ml /ml /ml
2 1 1 1 32 1
2 1 1 1 32 1
2 1 1 1 32 2
2 1 1 1 32 0.25
2 1 1 1 32 0.25
2 1 1 1 32 0.25
2 1 1 2 16 0.25 0.25
2 1 1 2 64 64 64
2 1 1 2 64 16 0.25
2 1 1 2 16 16 2
2 1 1 2 64 64 32
2 1 1 2 64 64 2
2 2 1 1 4
2 2 1 1 16 4
2 2 1 1 16 4
2 2 1 1 4
2 2 1 1 16 2
2 2 1 1 32 0.25
2 2 1 2 128 32 32
2 2 1 2 4 4 1
2 2 1 2 128 128 32
2 2 1 2 128 16 8
2 2 1 2 128 32 16
2 2 1 2 128 0.5 0.5
2 3 1 1 0.25
2 3 1 1 0.5 0.25
2 3 1 1 0.5 0.25
2 3 1 1 1 0.5
2 3 1 1 0.5 0.125
2 3 1 1 0.5 0.125
2 3 1 2 4 0.5 0.5
2 3 1 2 4 4 4
2 3 1 2 4 4 0.125
2 3 1 2 4 4 2
2 3 1 2 4 4 4
2 3 1 2 4 4 4
2
2
2
2 1 2 2 32 32 32
 44
2 1 2 2 4 2 0.25
2 1 2 2 1 0.5 0.5
2 1 2 2 4 2 2
2 1 2 2 4 2 2
2 1 2 2 2 2 2
2 2 2 2 0.5 0.5 16
2 2 2 2 0.5 0.5 0.5
2 2 2 2 0.5 8 8
2 2 2 2 1 0.5 8
2 2 2 2 0.5 0.5 0.5
2 2 2 2 1 0.5 0.5
2 3 2 2 4 4 4
2 3 2 2 4 4 4
2 3 2 2 4 4 4
2 3 2 2 4 4 4
2 3 2 2 4 4 4
2 3 2 2 4 4 4

Key:
1 Amorolfine HCL
2 Terbinafine HCL
3 Ciclopirox olamine
4 Amorolfine HCL + Ciclopirox olamine
5 Amorolfine HCL + Terbinafine HCL
Drug 6 Terbinafine HCL + Ciclopirox olamine
Ciclopirox olamine ( C )
C in (A+C)
C in (C+T)

Fungi 1 Candida albicans

species 2 Trichophyton mentagrophytes

1 Broth microdilution test-single drug

2 Broth microdilution test-combination test-single drug test
Experimen Broth microdilution test-combination test-combination
t 3 drug
 45
APPENDIX 2:
Combination Drug Test
Dat
e
plat Drug Fungi
Studen e combinatio specie Experime MIC 100 MIC 80 MIC 50
t ID read n s nt /ml /ml /ml
0.25∶0.12
2 4 1 3 1∶4 0.25∶0.25 5
2 4 1 3 1∶2 0.5∶0.125 0.25∶0.5
0.25∶0.12
2 4 1 3 0.25∶4 4∶0.125 5
2 5 1 3 16∶2 16∶4 0.5∶2
2 5 1 3 128∶8 128∶4 0.5∶2
2 5 1 3 16∶2 4∶2 0.5∶2
2 6 1 3 16∶0.125 16∶0.25 0.5∶0.125
2 6 1 3 8∶4 32∶0.125 0.5∶0.125
128∶0.12
2 6 1 3 128∶4 5 16∶0.125
2
2
2
2 4 2 3 8∶0.125 8∶4 4∶0.125
2 4 2 3 8∶0.125 4∶0.125 0.25∶4
2 4 2 3 1∶0.125 0.5∶0.125 0.5∶2
2 5 2 3 0.5∶2 0.5∶64 0.5∶64
2 5 2 3 0.5∶2 0.5∶2 0.5∶2
2 5 2 3 0.5∶2 2∶2 2∶2
0.5∶0.12
2 6 2 3 5 0.5∶1 0.5∶0.125
0.5∶0.12
2 6 2 3 5 0.5∶0.125 4∶4
0.5∶0.12
2 6 2 3 5 4∶0.125 4∶1

Key:

4 Amorolfine HCL : Ciclopirox olamine

5 Amorolfine HCL : Terbinafine HCL

Drugs 6 Terbinafine HCL : Ciclopirox olamine

 46

1 Candida albicans
Fungi
species 2 Trichophyton mentagrophytes

Broth microdilution test-combination test- combination

Experiment 3 drug

APPENDIX 3: Geometric means

MIC 100 for A/A/T for C/T/C

C.albicans mean geomean mean geomean
A+C 0.8 0.6 3.3 3.2
A+T 53.3 32.0 4.0 3.2
T+C 50.7 25.4 2.7 1.3

MIC 100 for A/A/T for C/T/C

T.mentag mean geomean mean geomean
A+C 5.7 4.0 0.1 0.1
A+T 0.5 0.5 2.0 2.0
T+C 0.5 0.5 0.1 0.1

MIC 80 for A/A/T for C/T/C

C.albicans mean geomean mean geomean
A+C 1.6 0.8 0.2 0.2
A+T 49.3 20.2 3.3 3.2
T+C 58.7 40.3 0.2 0.2

MIC 80 for A/A/T for C/T/C

T.mentag mean geomean mean geomean
A+C 4.2 2.5 1.4 0.4
A+T 1.0 0.8 22.7 6.3
T+C 1.7 1.0 0.4 0.3

MIC 50 for A/A/T for C/T/C

C.albicans mean geomean mean geomean
A+C 0.3 0.3 0.3 0.2
 47
A+T 0.5 0.5 2.0 2.0
T+C 5.7 1.6 0.1 0.1

MIC 50 for A/A/T for C/T/C

T.mentag mean geomean mean geomean
A+C 1.6 0.8 2.0 1.0
A+T 1.0 0.8 22.7 6.3
T+C 2.8 2.0 1.7 0.8

Appendix 4:
Disk Diffusion silver nanoparticles
Disk
Fungu Conten numbe 1st reading zone of 2nd reading zone of
s Drug t r inhibition in cm inhibition in cm
2 1 / 1 0 0
2 1 / 2 0 0
2 1 / 3 0 0
solvent
2 control / 4 0 0
2 2 / 1 0 0
2 2 / 2 0 0
2 2 / 3 0 0
solvent
2 control / 4 0 0
2 3 / 1 0 0
2 3 / 2 0 0
2 3 / 3 0 0
solvent
2 control / 4 0 0
2 4 / 1 0 0
2 4 / 2 0 0
2 4 / 3 0 0
solvent
2 control / 4 0 0
2 5 / 1 0 0
2 5 / 2 0 0
2 5 / 3 0 0
solvent
2 control / 4 0 0
2 6 / 1 0 0
2 6 / 2 0 0
2 6 / 3 0 0
solvent
2 control / 4 0 0
2 7 / 1 0 0
 48
2 7 / 2 0 0
2 7 / 3 0 0
solvent
2 control / 4 0 0
2 8 / 1 0 0
2 8 / 2 0 0
2 8 / 3 0 0
solvent
2 control / 4 0 0
2 9 / 1 0 0
2 9 / 2 0 0
2 9 / 3 0 0
solvent
2 control / 4 0 0
2 10 / 1 0 0
2 10 / 2 0 0
2 10 / 3 0 0
solvent
2 control / 4 0 0
2 11 / 1 0 0
2 11 / 2 0 0
2 11 / 3 0 0
solvent
2 control / 4 0 0

Key:
Fungus 2 Candida albicans

1 RB429ii
2 RB434ii
Drug 3 RB433iii
4 RB433i
5 RB431i
6 RB433ii
7 RB430i
8 RB430iii
9 RB430ii
10 RB429iii
11 RB429i

1 /
Content 2 /

1st
reading after 24 hours
 49
2nd
reading after 48 hours

1 Disk one
2 Disk two
Disk 3 Disk three
number 4 Solvent control

Appendix 5:
Disk Diffusion T.mentagrophytes
Disk 1st reading zone of inhibition
Fungus Drug Content number in cm
1 1 1 1 0
1 1 1 2 0
1 1 1 3 0
solvent
1 control 1 4 0
1 2 1 1 0
1 2 1 2 0
1 2 1 3 0
solvent
1 control 1 4 0
1 3 1 1 1
1 3 1 2 1
1 3 1 3 1
solvent
1 control 1 4 0
1 3 2 1 2.5
1 3 2 2 2.5
1 3 2 3 0
solvent
1 control 2 4 0

Key:

Fungus 1 Trichophyton mentagrophytes

1 Amorolfine HCL
2 Terbinafine HCL
Drug 3 Ciclopirox olamine
 50

1 5 /disk
Content 2 50 /disk

1st
reading after 168 hours

1 Disk one
2 Disk two

Disk 3 Disk three

number 4 Solvent control