1. Faseela TS/71/January 2012 batch Pre-Colloquium
Biofilm formation and efflux mediated azole resistance among clinical isolates of Candida albicans - an in vitro study  
Faseela TS/71/January 2012 batch
Pre-Colloquium
Name of the student:
Mrs. Faseela TS
Dept. of Microbiology
YMC
Guide:
Dr. Raja Gopal K
Professor & HOD
Dept. of OBG, YMC

2. Introduction
The genus Candida have exploded into prominence in recent years as opportunistic and nosocomial pathogen
C. albicans is the most common species
Biofilm formed on biomaterials is a risk factor
It leads to persistence of infection and increased drug resistance
Azoles are the primary drugs in the management Candidiasis
There is a spurt of resistance to azoles recently in Candida including C. albicans

3. Azole resistance is mainly due to increased drug efflux by efflux pumps
The ATP-binding cassette (ABC) and major facilitator super family (MFS) transporters are important efflux pumps
Azoles and Rhodamine 6G are the substrates for ABC transport pumps coded by CaCDR1 and CaCDR2 genes.
The drugs which interfere with efflux can potentiate the antifungal activity of azoles

4. Review of Literature
Candida is fourth commonest cause of blood stream infections (Todd McCarty et al., 2015)1
The fifth most common cause of nosocomial infection and fourth among blood stream pathogens according to CDC-NHSN report (Dawn M et al.,2013)2
In India, incidence of candidiasis varies from 5.7% to 18% (Giri S et al.,2012)3
An increase in the incidence of candidiasis has also been reported from different parts of the world

5. Until recently C. albicans was the most common species
There is emergence of Non Candida albicans species dramatically in the last 30 years
C. albicans still remains as most prevalent species according to ARTEMIS and SENTRY reports (yapar et al.,2014) 4
ARTEMIS DISK global survey shows a decrease in prevalence of C. albicans from 70.9% ( ) to 62.9% ( ) with increase in C. tropicalis (from 5.4% to 7.5%) (Pfaller et al.,2010) 5

6. C. albicans is reported as predominant species from Maharashtra (Rachana et al., 2016, Aher et al., 2014), Punjab (Sheevani et al.,2013) and Puduchery (Mohan et al., 2013 ) 6,7,8,9
There are reports showing C. tropicalis as the most common species from Malaysia (Kee Peng et al.), Delhi (Ravinder et al.,2014 ) and Tamil Nadu (Binesh et al.,2011) 10,11,12
Most of the studies from Karnataka shows C. albicans as the most common species with a range of 39% to 65%, followed by C. tropicalis (Roopa et al.,2015, Amar et al., 2014, Mohandas et al., 2011) 13,14,15

7. However, C. tropicalis was predominant in a study from Mysore (Tejashree et al.,2014) 16
A study report from Mangalore reported a prevalence 47% for C. albicans followed by C. tropicalis (30%). (Shivanand et al.,2011) 17
Biofilm formation is an important virulence factor, leads to increased mortality
Mortality rate of 51.2% in the biofilm forming C. albicans when compare to 31.7% in nonbiofilm-forming group (Tumbarello et al., 2012) 18

8. Data on biofilm formation from different countries ranging from 36% to 100% (Jasim et al.,2016 , Zarei et al.,2014 , Villar Vidal et al., 2011) 19,20,21
In India, it is 40-50% as reported from different states (Agwan et al., 2015, Ravinder Kaur et al.,2014, Sahar Ali et al.,2013,) 22,11,7
However, reports from Karnataka shows a range of 40-62% (Chaithanya et al., 2014, Saroj et al.,2012, Mohandas et al.,2011) 23,24,25
A study from Mangalore reported 55%. (Udayalaxmi et al.,2014) 26

9. WHO report on antimicrobial resistance surveillance published 2% (Korea, Denmark)-50%(South Africa) azole resistance among C. albicans (WHO,2014) 27
Studies from India report azole resistance ranging from 0% to 63% (Kumar et al., 2016, Mohan et al., 2016, Sheevani et al.,2013 ) 8,9,78
Reports published from Karnataka shows a range of 0-38% (Tejashree et al.,2015, Chaithanya et al.,2014, Roopa et al.,2015, Amar et al., 2014, Shivanand et al., 2011) 13,14,16,17,23

10. An increased glucose-induced efflux of Rhodamine 6G is reported in experimental FLC-resistant C. albicans (Lan Yang et al.,2008, Mishra et al.,2007) 29
Azole resistant C. albicans are shown to over express genes encoding drug efflux pump proteins (Monika et al.,2011, Oscar Marchetti et al.,2000) 30,31
The agents that prevent efflux may be useful for management of azole resistant C. albicans
MET inhibitors such as cyclosporine A and ibuprofen showed potent antifungal effect when used in combination with azole (Marchetti et al., 2000, Monika Sharma et al., 2005) 30,31

11. Social relevance of the study
This study will show the distribution pattern of different species of Candida in candidiasis
Status of drug resistance among C. albicans
Antifungal susceptibility test data of various drugs may lead to development of guidelines for effective treatment of candidiasis
The study on biofilm formation will contribute towards the development of preventive strategies and patient management

12. The study on in vitro efflux mechanism of drug resistance will be the base for developing new antifungal drugs
The study on combination of azole with cyclosporine and ibuprofen might open a new therapeutic approach for the management of drug resistant C. albicans infections

13. Aim
To study biofilm formation, azole resistance and energy dependent efflux among Candida albicans isolated from clinical specimens and to study antifungal effect of azole-efflux inhibitor combinations on them

14. Objectives To identify clinical isolates of Candida species
To study biofilm formation in C.albicans
To determine in vitro antifungal susceptibility and minimum inhibitory concentration of antifungal agents used for parenteral, oral and topical applications
To study in vitro energy dependent efflux among azole resistant C.albicans
To study the antifungal effect of azole- efflux inhibitor combinations on those strains

15. Research methodology Materials and Methods:
Experimental design: Prospective observational experimental study
Sample size: Two hundred Candida species isolated from clinical specimens
Place of study: Department of Microbiology, Yenepoya Medical College
Inclusion criteria: Candida isolated from patients with suspected candidiasis
Exclusion criteria: Polymicrobial growth from normally sterile specimen

16. Cultural characteristics on SDA
1. Isolation and identification of Candida It was done by using standard procedures 33
Cultural characteristics
on SDA
Fig.2:Candida species on SDA.
Gram’s stain
Fig.2: Candida on Gram’s stain
Urea hydrolysis
Fig.3:Gram’s stain

17. Cultural characteristics on Hichrom Candida agar
Germ tube test
Fig.4:HiChrom agar with Candida species
Growth at 420 C
Chlamydospore formation
Fig.5: Chlamydospore formation

18. Carbohydrate assimilation test Glucose, Maltose,
Sucrose, Lactose, Galactose, Melibiose, Cellibiose, Inositol, Xylose, Raffinose, Trehalose, Dulcitol
Fig.6: Sugar assimilation test
Carbohydrate fermentation test
Fig.7: Sugar fermentation test

19. 2. Biofilm Formation Microtiter plate assay
Method: Christensen et al. with minor modifications 34
Stained by 0.5% crystal violet
Destained by using 95% ethanol
Fig.8: Biofilm formation on microtiter plate
Quantification by measuring OD value at a wavelength of 580 nm

20. OD value measured by Multimode microtiter plate reader (FLUOstar Omega - Bitotron Heatlh care
Interpreted as strong biofilm producer, moderate biofilm producer, weak biofilm producer and no biofilm producer
Fig.9: Quantification of biofilm by Multimode microtiter plate reader

21. 3. Antifungal susceptibility test for C. albicans
- Disk diffusion method CLSI M44-A2 35
Mueller Hinton Agar with 2% dextrose and 5 µg/ml methylene blue was used
Antifungals: fluconazole, itrakonazole, ketoconazole, miconazole, voriconazole and amphotericin B
Fig.10: Antifungal susceptibility test

22. susceptible dose dependent
Table 1:Interpretation table
Antifungal
Potency
Zone diameter (mm) R ≤
SDD S ≥
Fluconazole*
25 µg 14
15-18 19
Itraconazole
10 µg 9
10-15 16
Ketoconazole
15 µg 22
23-29 30
Voriconazole*
1 µg 13
14-16 17
Miconazole
50 µg 11
12-19 20
Amphotericin B
10-14 15
Interpreted as:
susceptible
susceptible dose dependent
resistant
S-susceptible, SDD- susceptible dose dependent, R- resistan
* As per CLSI M44A2

23. Microbroth dilution method CLSI M 27-A3
Medium used: RPMI 1640 (with glutamine, without bicarbonate) 35
Antifungals used: fluconazole, itraconazole ketoconazole and voriconazole
Fig.11:MIC detection on microtiter plate
Dilutions:
Fluconazole to 64μg/mL
Ketoconazole & voriconazole to 16μg/mL
Fig.12: Microtiter plate fluconazole resistant C. albicans

24. Stock solution preparation
Table 2. Scheme for Preparing Dilutions of Water Soluble Antifungal Agents

25. Stock solution preparation
Table 3: Scheme for Preparing Dilution Series of Water-Insoluble Antifungal Agents

26. Susceptible dose dependent resistant
Reference strain:
C. albicans ATCC
Table 4: MIC Interpretation table
Antifungal agents
MIC breakpoints
S (g/ml)
SDD (g/ml)
R (g/ml)
Fluconazole*
≤ 8.0
16-32
≥ 64
Voriconazole*
≤ 1.0
2.0
≥ 4.0
Ketoconazole
≤ 0.12
0.25 to 0.5
≥ 1
Itraconazole
Interpreted as :
Susceptible
Susceptible dose dependent
resistant
S-susceptible, SDD- susceptible dose dependent, R- resistant

27. 4. Energy dependent efflux of among azole resistant C.albicans
By using ABC transport substrate Rhodamine 6G
Method described by Kevin et al. is used 37
Glucose is supplied as source of energy

28. C. albicans grown in YPD broth
1 ml was taken in to 100 ml fresh YPD
37°C
4 hrs
Centrifuged at 3000xg for 5 min
Washed twice with PBS
Suspended in 10 ml PBS buffer

29. Candida in 10 ml PBS buffer
I hr
Added Rhodamine 6 G (10 M)
30 min
37°C
Added glucose
Added PBS
37°C for 30 min
Removed 1 ml
Removed 1 ml
Removed 1 ml
Centrifuged at g for 1min
Measured absorbance of supernatant at 527nm

30. Glucose induced efflux of Rhodmine 6 G:
Fig.13: Efflux study
Fig.14: OD value measurement by
Multimode microtiter plate reader
Glucose induced efflux of Rhodmine 6 G:
OD Value of R6G in the supernatant of glucose supplemented sample - Control sample

31. 5. Antifungal activity of azole–efflux inhibitor combinations on azole resistant C.albicans:
Done by agar diffusion by as described by Marachetti et al 35
Cys
Cyclosporine A and ibuprofen was tested in combination with azole by agar dilution method Ib
Medium used: YEPD agar with different concentrations of fluconazole, itraconazole and voriconazole
Fig. 15: Inhibition of C. albicans growth by Cyclosporine A and Ibuprofen on YEPD agar containing azole

32. Cyclosporine A disk of 0.625µg/ml
Table 5:Interpretation table
Low
Intermediate
High 
<1 mm/μg
≥1 to <10 mm/μg
≥10 mm/μg
Cyclosporine A disk of 0.625µg/ml
Ibuprofen disk of
50 μg/mL

33. Data management and statistical analysis
By using SPSS software
Chi-square test ant fishers exact test was used for comparison of variables
P values of <0.05 was considered statistically significant

34. Results

35. 1.1 Demographic distribution of candidiasis
Distributed equally between male and female
Fig. 16: Distribution of various samples among male and female

36. 1.2 Distribution of sample in different age group
Common among the age group of yrs
In case of vaginal discharge, the major age group was yrs
Fig.17:Distribution of sample among different age group

37. 1.3. Predisposing factors/ underlying conditions
Table 4: Risk factors among patients with candidiasis
Risk factors/Underlying conditions
No. of patients %
Diabetes Mellitus 35 15
Pregnancy 44 23
CKD/Renal failure 7 3
COPD 6
CLD 2 1
Pulmonary tuberculosis
HIV
0.5
Hepatitis B
Malignancy
Surgery 12 5
Antibiotic Rx
106 47
Steroid Rx
Septic shock
Preterm
ICU stay

38. 1.4. Species distribution
Table 5: Distribution of Candida species in various clinical specimen
Specimen
Vaginal discharge
Oral
Pus
Blood
Urine
Nail
Body fluids
Ear
Skin
Tissue
Total %
C. albicans 36 29 10 5 3 -  2 98
49.5
C. tropicalis 12 14 4 1 2  - 50 25
C. krusei 6
C. parapsilosis 11
C. glabrata 7
C. dubliniensis
C.guillermondii
C. lambica
C. kefyr
C. lusitaneae
Candida species 56 37 32 8
200
100
28%
18.5%
16%
7.5% 4%
3.5% 3% 2%

39. Among 56 isolates from vaginal discharge, C
Among 56 isolates from vaginal discharge, C. albicans was the most common isolate (64%) followed by C. tropicalis (21%)
Among 37 isolates from from oral specimen, C. albicans was the most common isolate (78%) followed by C. tropicalis (14%)
In 32 blood specimens, C. tropicalis was the most common species (44%) followed by C. albicans (16%)
Prevalence C. tropicalis and C. albicans equal in (30%) in 10 pus samples

40. 1.5. Comparison of C. albicans & NAC in mucocutaneous and invasive infections
C. albicans was more prevalent among mucocutaneous infections when compare to invasive infections (p value‹0.0001)
Among Candida causing invasive infections, C. tropicalis was the predominant species (34%)

41. 2. Biofilm formation among C. albicans
Among 99 C. albicans tested for biofilm formation 53 were biofilm producers

42. 2. 1. Biofilm formation among C
2.1. Biofilm formation among C. albicans isolated from various specimens
Table 6: Biofilm forming C.albicans among various clinical specimen
Specimen
Negative
Weak
Moderate
Strong
Total
Oral specimen 16 7 1 5 29
Vaginal discharge 10 2 8 36
Pus 6 -
Nail
Urine
Blood 4
Ear discharge 3
Fluids  -
Tissue
 Total 46 26 20 9  %
47%
26% 7%
20%
100%

43. 2. 2. Comparison of biofilm forming C
2.2. Comparison of biofilm forming C. albicans isolated from sample representing mucocutaneous and invasive infections
Table 7: Comparison of biofilm producers and non biofilm producers among mucocutaneous and invasive infections
Among 36 C. albicans isolated from vaginal discharge, 56% were biofilm producers
Mucocuta-neous infections
Invasive infections
Biofilm producer 37 16
Non biofilm producer 33 13
Among 29 C. albicans isolated from oral specimen, 45% were biofilm producers
Among 10 C. albicans isolated from pus, 40% were biofilm producers
There is no association between biofilm formation and type of infection
P value equals

44. 3. Antifungal susceptibility pattern of C. albicans
Table 8: Antifungal susceptibility pattern of C. albicans
Fluconazole
Itraconazole
Ketoconazole
Voriconazole
Miconazole
Amphotericin B S
SDD R
Blood 5 -  -
Ear 2 1 3
Oral 22 4 19 6 25 27 29
Pus 10
Fluids
Tissue
Urine
Nail
Vaginal 32 33 36
Total 85 9 84 11 91 8 97 99 % 86 92 98
100
S-susceptible, SDD- susceptible dose dependent, R- resistant

45. 3. 3. Comparison of azole resistance among C
3.3. Comparison of azole resistance among C. abicans causing mucocutaneous & invasive infections
Table 9: Comparison of azole resistance among C. abicans causing mucocutaneous infections & invasive infections
% of resistance
Fluconazole
Itraconazole
Ketoconazole
Voriconazole
Mucocutaneous infections 11 13 10
Invasive infections 6 12 3
There is no significant association between fluconazole resistance among  C. abicans causing mucocutaneous infections and invasive infections
p value=

46. 3.4. Correlation between biofilm formation and azole resistance
Among biofilm producers, 29% (15) isolates were resistant to one of the azole group of antifungals
In case of non-biofilm producers it was 21%
There is no association between biofilm formation and azole resistance.
P value equals ,which is considered to be not statistically significant

47. 4. Energy dependent efflux among azole resistant C. albicans
Table 10: Glucose induced Rhodamine 6 G efflux among azole resistant C. albicans
Azole resistant
C. albicans
Efflux negaitive
Efflux positive
Total
No. 8 12 20 % 40 60
100

48. 5. The antifungal effect of azole-efflux inhibitor combinations on energy dependent efflux mediated azole resistant strains
i. Combination of Cyclosporine A with azoles
Table 11: Combination of Cyclosporine A with azoles
Fluconazole
Itraconazole
Ketoconazole
Voriconazole
Antifungal
( µg/ml)
8 , 16, 32 & 64
1,2,4 & 8
2,4,8 &16
Cyclosporine A
(µg/ml)
0.625
No. of isolates tested 05
Effect
Synergism

49. ii. Combination of ibuprofen with azoles
Table 12: Combination of ibuprofen with azoles
Fluconazole
Itraconazole
Ketoconazole
Voriconazole
Antifungal
( µg/ml)
8 , 16, 32 & 64
1,2,4 & 8
2,4,8 &16
Cyclosporine A
(µg/ml) 50
No. of isolates tested 05
Effect
Synergism

50. Discussion 1.1. Demographic distribution of candidiasis
There is no association between candidiasis and gender in present study
There are previous study reports showing more prevalence in female (Shivanand et al. 2011) 17 and male (Amar et al.,2014, Ravinder et al., 2014) 11,14
The most commonest age groups are of years
Previous study shows wide difference in this aspect
Shivanand et al. reported years as common age group17

51. 1.2. Predisposing factors and underlying conditions
Broad spectrum antibiotic treatment, pregnancy and diabetes mellitus were the important risk factors
It is comparable with previous reports (Ravinder et al., 2014, Amar et al., 2014, Shivanand et al.,2011) 11,14,17

52. 1.3. Species distribution
C. albicans was major species followed by C.tropicalis
Comparable with previous studies (Rachana et al., 2016, Roopa et al., 2015, Udayalaxmi et al.,2014, Shivanand et al.,2011)6,14,17,26
C. tropicalis is the predominant isolate in a few studies (Kumar et al.,2016, Tejashree et al.,2014, Binesh et al.,2011)12,16,28

53. 5. Biofilm formation Biofilm producers in the present study were 50%
Comparable with previous studies (Mohandas et al., Kumar et al.,2016)25,28
A higher prevalence of % is reported previously (Jasim et al.,2016, Chaithanya et al.2014, Zarei et al.,2014,)119,20, 23

54. 6. Antifungal susceptibility pattern
Antifungal sensitivity in present study was 86%, 85%, 86% and 92% for fluconazole, Itraconazole, ketoconazole and voriconazole respectively
Similar to previous studies (Binesh et al.,2011, Mondal et al. 2013, Yashwanth et al.,2013) 12,38,39
But, there are also a few reports of 100% sensitivity to azoles (Yotsabeth et al ., 2015, Tejashree et al.,2014, Sheevani et al.,2013) 8,16,40
A higher resistance of more than 50% also reported previously (Mohan et al.,2016 )25

55. 7. Rhodamine efflux among azole resistant C. albicans
Increased Rhodamine efflux was seen among 60% of the azole resistant C. albicans when energy was supplied
Increased efflux of Rhodamine 6G is reported previously also among azole resistant C. albicans (Mishra et al.,2007, Lan Yang et al.,2008, Jin Yan et
al .,2015) 29,41,42

56. 7. Azole –efflux inhibitor combination on azole resistant C
7. Azole –efflux inhibitor combination on azole resistant C. albicans with Rhodamine efflux
All the azole resistant C. albicans positive for glucose induced Rhodamine efflux became susceptible to azole when it combine with Cyclosporine A and ibuprofen
Similar reports published by (Marchetti et al., 2000, Monika et al., 2005) 30,31
Pina Vaz et al reported that eight of the 12 strains of Candida studied showed synergic activity when fluconazole combined with ibuprofen (Pina Vaz et al., 2005)43

57. Conclusions
Even though there are many changes in the epidemiology, C. albicans remains as major pathogen of Candidiasis in this area
There is an emergence of Non albicans Candida species such as C. tropicalis, C. krusei, C. parapsilosis, C. glabrata, C. dubliniensis and C. guillermondii
It emphasizes the need to speciate Candida isolates routinely in diagnostic laboratories
C. albicans still shows good susceptibility to Amphotericin B.

58. However, up 15% of them show resistance to azoles that makes routine antifungal susceptibility test and periodic surveillance mandatory
biofilm formation seen in 50% of the C. albicans highlight importance of practicing effective patient management in the hospital
Increased efflux is the one of the major mechanisms of azole resistance
Efflux inhibitors such as ibuprofen and cyclosporine A in combination with azoles could be the possible treatment option for azole resistant C. albicans

59. Highlights of the study
C. albicans is the major species causing candidiasis
C. tropicalis is predominant in blood and pus sample
Azole resistance and biofilm formation are problems associated with C. albicans
More than half of the C. albicans are with increased efflux indicate it as the one of the major mechanisms of azole resistance
Combination studies highlight the effectiveness of ibuprofen and cyclosporine A along with azoles against azole resistant efflux positive C. albicans

60. Limitations of the study
Detection of azole efflux was not done specificlly
Molecular method for detection of efflux was not attempted
Scope for future work
Rapid method for detection of azole resistant C. albicans by using molecular method will be useful for management of patient
Biofilm formation has to be evaluated by using materials which are commonly used on patients in the hospital

61. The structural resolution of efflux pumps may help in rational drug design
Detailed study on azole- efflux inhibitor combination may be useful to develop treatment strategies for azole resistant Candida
Effectiveness of the combinations has to be evaluated in vivo

62. Layout of thesis SI. No. Content Page No. ABSTRACT I LIST OF TABLES iv
LIST OF FIGURES Vi
LIST OF APPENDICES
vii
CHAPTER 1 INTRODUCTION
1.1
General introduction 1
1.1.1
Pathogenecity of Candida 3
1.1.2
Antifungal agents and drug resistance among C. albicans 4
1.1.3
Mechanisms of azole resistance in C. albicans 5
1.1.4
Prevention of antifungal resistance 6
1.1.5
Management of azole resistant C. albicans
1.2
Aim of the study 9
1.3
Objectives of the study
1.4
Social relevance of the study 10

63. CHAPTER 2 REVIEW OF LITERATURE 12
2.1
Introduction to fungi
2.2
History
2.3
Taxonomy 13
2.4
Morphology of Candida 14
2.4.1
Dimorphism
2. 4.2
Cell wall structure 15
2. 5
Epidemiology and Ecology 16
Trends in species distribution 17
International scenario
National scenario 18
Distribution pattern of Candida species from different anatomical sites 20
2. 6
Immunity 21
Innate immune mechanisms
2.6.2
Humoral immunity 23
2.7
Virulence factors
2.7.1
Adherence
2.7.3
Secreted hydrolytic enzymes 24
2.7.4
Hemolysin

64. 2.7.5
Candida lipases 24
2.7.6
Integrin receptor
2.7.7
Dimorphis
2.7.8
Quorum sensing 25
2.7.9
Thigmotropism
2.7.10
Phenotypic switching
2.7.11
Evasion to the host immune responses 26
2.7.12
Biofilm formation
Methods of quantification of biofilm 29
Trends in biofilm formation among C. albicans 30
International scenario
National scenario
2.8
Risk factors for invasive candidiasis 31
2.9
Candida infections
2.9.1
Mucocutaneous infections
Cutaneous infections
Nail infections

65. 2.9.2
Mucosal infections 33
Chronic mucocutaneous candidiasis
Oral candidiasis
Candidial vulvovaginitis 34
2.9.3
Invasive candidosis
Candidemia
Oesophageal candidiasis
Candiduria 35
Other infections caused by Candida
2.10
Laboratory diagnosis of candidiasis
2.10.1
Direct examination 36
2.10.2
Isolation
CHROM agar Candida
2.10.3
Germ tube test 37
2.10.4
Chlamydospore formation
2.10.5
Tests for detection of antibodies
2.10.6
Tests for detection of antigens

66. 2.10.7
Molecular techniques 38
2.11
Antifungal drugs
2.11.1
Classification of Antifungal agents 39
2.11.2
Polyenes 40
2.11.3
Azoles
2.11.4
Echinocandins 41
2.11.5
Antimetabolite
2.11.6
Nikkomycin 42
2.11.7
Allylamine
2.11.8
Mechanisms of azole resistance
2.11.9
Antifungal drug susceptibility testing 43
Trends in antifungal resistance among clinical isolates of C. albicans 44
International scenario
National scenario
2.12
Efflux 45
2.12.1
Classes of efflux pumps
MFS Transporters 46

67.
ABC Transporters 46
2.12.2
Efflux mediate azole resistance among C. albicans
Efflux pump mediated drug resistance as a stress response 48
2.12.3
Overcoming efflux-mediated antifungal drug resistance 49
2.13
Different targets and new therapeutic approaches
2.13.1
Efflux inhibitors and azole susceptibility 50
2.13.2
Methods of studying drug combinations 52
CHAPTER 3 METHODOLOGY
3.1
Materials and Methods 53
3.1.1
Study setting
3.1.2
Study population
3.1.3
Ethical clearance
3.1.4
Clinical specimens- Inclusion criteria
3.1.5
Clinical specimens- Exclusion criteria
3.2
Isolation, identification and speciation of Candida
3.2.1
Colony morphology on Sabouraud’s dextrose agar
3.2.2
Grams staining 54

68. 3.2.3
Urease test 54
3.2.4
Hicrome Candida agar morphology 55
3.2.5
Germ tube test
3.2.6
Cornmeal agar culture
3.2.7
Carbohydrate assimilation tests 57
3.2.8
Sugar fermentation tests 58
3.2.9
Growth at 42°C 59
3.3
Biofilm Formation
3.3.1
Preparation of inoculum 60
3.3.2
Procedure
3.3.3
Interpretation of result
3.4
Antifungal susceptibility test 61
3.4.1
Disk diffusion method (M44A2)
Preparation of McFarland 0.5 standard 63
3.4.2
Micro broth dilution method (CLSI M27-A3)
Antifungal agents
Preparation of antifungal stock solutions
Medium 64

69.
Preparation of antifungal dilution 64
Inoculum Preparation 66
Procedure 67
Interpretation of results
3.5
Energy dependent efflux among azole resistant C.albicans 68
3.5.1
Preparation of inoculum
3.5.2
3.6
The antifungal effect of azole-efflux inhibitor combinations on azole resistant strain 69
3.6.1
3.6.2
Efflux inhibitor agents
3.6.3
Preparation of efflux inhibitor agents stock solutions 70
3.6.4
Preparation of drug dilutions
3.6.5
Medium used
3.6.6
3.6.7
Interpretation of the result 71
CHAPTER 4 RESULTS
4.1
Clinical specimens 72
4.2
Demographic distribution of candidiasis
4.3
Predisposing factors 74

70. 4.4
Species distribution 75
4.4.1
Species distribution in vaginal discharge 76
4.4.2
Species distribution in oral specimen 77
4.4.3
Species distribution in pus
4.4.4
Species distribution in blood 78
4.4.5
Prevalence of C. albicans in skin & mucous membrane 79
4.4.6
Prevalence of C. albicans in exudate & body fluids 80
4.4.7
Comparison of C. albicans & NAC in mucocutaneous and invasive infections
4.5
Biofilm formation among C.albicans 81
4.5.1
Biofilm formation among C. albicans isolated from vaginal discharge 82
4.5.2
Biofilm formation among C. albicans isolated from oral swab
4.5.3
Biofilm formation among C. albicans isolated from pus 83
4.5.4
Comparison of biofilm forming C. albicans isolated from sample representing mucocutaneous and invasive infections 84
4.6
Antifungal susceptibility pattern of C. albicans 85
4.6.1
Azole susceptibility among C. albicans isolated from samples representing mucocutaneous infections 86
4.6.2
Azole susceptibility among C. albicans isolated from samples representing invasive infections 87
4.6.3
Comparison of azole resistance among C. abicans causing mucocutaneous infections & invasive infection

71. 4.6.4
Correlation between biofilm formation and azole resistance
4.7
Energy dependent efflux among azole resistant C.albicans
4.8
The antifungal effect of azole-efflux inhibitor combinations on azole resistant strains
4.8.1
Combination of Cyclosporine A with azoles 90
Combination of Cyclosporine A with fluconazole
Combination of Cyclosporine A with itraconazole
Combination of Cyclosporine A with voriconazole 91
4.8.2
Combination of ibuprofen with fluconazole 92
Combination of ibuprofen with itraconazole
Combination of ibuprofen with voriconazole 93
CHAPTER 5 DISCUSSION 94
5.1
Demographic distribution of candidiasis
5.2
Predisposing factors and underlying conditions among patients 95
5.3
Species distribution
5.4
Biofilm formation 99

72. 5.5
Antifungal susceptibility pattern
100
5.6
Efflux
101
5.7
Combination approach
103
CHAPTER 6 SUMMARY AND CONCLUSIONS
106
6.1
A concise report of the work done
6.2
Findings of the study
6.3
Logical analysis presented in the discussion
107
6.4
Conclusion and Limitations of the study
6.5
Future prospects and suggestions
108
REFERENCES
109

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87. THANK YOU