CHAPTER.4

ROLE OF FLUCONAZOLE IN WOUND HEALING

Introduction

Diabetes has become the most common cause for non-traumatic amputation of lower

extremities all over the world (5, 161). It cause severe social and economic burden,

mental and psychological misery, diminished quality-of-life, increased morbidity and

mortality of people with diabetes across the world (2). Polymicrobial infections is

found to be the single most precursor to about 85% of the amputations among patients

with diabetic lower extremity wounds (DLWs) (111, 143). Despite the therapy with

antibiotics and surgical debridement of slough, wound healing is either impaired or

delayed even up to 18 weeks among diabetic patients (65, 66, 109). Several

pathogenic abnormalities, ranging from disease-specific intrinsic flaws in blood

supply, angiogenesis, and matrix turnover to extrinsic factors such as infections and

continued trauma, contribute to failure to heal DLWs (44).

It is reported that biofilm caused by common pathogens such as Staphylococcus sp

and Pseudomonas sp are not only associated with increased morbidity and mortality

but also contribute significantly to the emergence and dissemination of antibiotic

resistance (89). Specific interaction among microorganisms belonging to different

domains such as bacteria and fungi would communicate among microorganisms by

physical interactions or signals and induce biosynthesis genes (125). Our earlier study

showed mixed bacterial and fungal infections in 21.4%; and fungal infection alone in

5.8% of patients with DLWs (32). It is also known that toe web dermatophyte

infection provides a hospitable niche for subsequent colonization by bacteria (Figure

4.1 to 4.3) (70, 84).

Figure 4.1 Inter-digital fungal infection

Figure 4.2. Secondary bacterial infection on the inter-digital fungal infection

Figure 4.3. Gangrenous 2nd

toe and loss of 3rd

toe following secondary bacterial

infection.

In spite of these evidences on mixed bacterial and fungal infections in DLWs, the

current clinical practice is targeted only against bacterial infections and but not

against fungal infections as well. We speculate that fungal infections may have a

significant role in delaying wound healing and the morbidity of diabetic patients.

Aims: To test the hypothesis that fluconazole plus standard care for fungal infection

in DLWs is superior to standard care alone.

Patients and Methods:

Study design. This 34 week, randomized, case-controlled, open label, parallel arm

study was conducted to demonstrate superiority of fluconazole plus standard care over

standard care alone for deep seated fungal infections in DLWs. The study was

approved by the Institutional Ethics Committee, AIMSRC, which is in accordance

with the Declaration of Helsinki, Good Clinical Practice guidelines, and local laws

and regulations.

Sample size. As there were no available studies on the effect of fluconazole in

treating fungal infections of DLWs, we used our pilot study results for estimating

sample size. In the pilot study, the percentage wound reduction at week 2 was taken

as the reference point. The percentage wound reduction was 40.7 for treatment arm

and 22.5 for the control arm; and the effect size was 0.83390. With 95% confidence

interval and a power of 80, the sample size (n) was calculated as 48 (24 in each

group).

Patients. We included patients with Type 2 Diabetes, ages between 18 and 75 years,

having an open wound on lower limb, with a wound size >/=4cm2, deep tissue

showing fungal & bacterial culture positivity, fungi must be susceptible to

fluconazole, ankle brachial index (ABI): >/=0.7, vibration perception threshold

(VPT): >25, transcutaneous partial pressure of oxygen (TcPO2): >25mm Hg, serum

aspartate aminotransaminase (AST) and alanine aminotransferase (ALT): <3 times

upper limit of normal value and serum creatinine: <1.5 mg/dl. Fungal species were

identified morphologically and using ID32C strips (32, 120). Antifungal susceptibility

testing for yeasts was done with ATB Fungus-3 strips (141). Bacterial isolates were

identified by standard biochemical tests and susceptibility testing was performed as

per CLSI guidelines. All the study subjects were operated under either local or spinal

anaesthesia in the sterile operation theater. Written informed consent was obtained

from all the patients during enrollment.

We excluded pregnant and nursing mothers or patients with an HbA1c >14% or with

evidence of osteomyelitis over lower limb. Osteomyelitis was ruled out clinically

(probing to bone) and with nuclear scan. The subjects who were on anti-fungal

therapy or steroids or immunosuppressive or chemotherapeutic agents or radiotherapy

in past 6 months were also excluded from the study.

Randomization. A total of 75 patients were randomly assigned either to the standard

care arm (C) alone or to standard care plus fluconazole (T) arm in 1:1 ratio using

block (size of ten) randomization technique.

Treatment and follow-up. Patients in both study arms received standard care

(surgical debridement + culture specific antibiotics + offloading + glycemic control)

according to the local protocols. Necrotic tissues were tissues were removed in the

sterile operation theater and deep tissue from the wound-bed was cultured for bacteria

and culture specific antibiotics were given. The wounds were dressed only with

normal saline. We did not use other wound care products for the study subjects.

Offloading was done using modified footwear or wheel chairs or walkers. Blood

sugars were controlled with oral hypoglycemic agents and Insulins. T arm received

fluconazole 150 mg/day in addition to standard care till outcome measures were met.

In order to ensure treatment compliance, tablet fluconazole was given free of cost for

all the patients in the T arm. A trained Podiatrist, who was blinded about the treatment

given to each subject, measured the wound size. In order to measure the complete

dimension of the DLW, a sterile, thin, polythene sheet was placed over the wound

base and another sterile polythene sheet was wrapped over that and wound margins

were traced with a marker (figure 4.4). The maximum length and breadth

(perpendicular to each other) were measured to calculate the wound surface area

(WSA). All the patients were followed up every 2 weeks for 34 weeks or till they

meet the treatment outcomes.

Figure 4.4. Tracing the wound with sterile transparent polythene sheet

Treatment compliance. Treatment compliance was measured every two weeks

during their follow-up visits. Patients were interviewed on the offloading and

fluconazole or antibiotics or OHA/Insulin intake during every follow-up visit and

their responses were recorded. Above 85% was considered as good compliance over

the study period.

Outcome measures/end points. We considered complete epithelialization or skin

grafting was considered as primary end point and the mean wound surface area as the

secondary endpoint. These end points were assessed clinically by a blinded podiatric

nurse.

Statistical analysis. The trial was designed to demonstrate superiority of fluconazole

plus standard care over standard care alone for treating deep seated fungal infections

in DLWs. Comparison of the wound surface area (WSA) between the two T and C

arms at baseline showed that the difference was statistically not significant, i.e the two

groups were comparable at baseline level on an average. The standard deviation of the

WSA at both the arms was high and the sample size was small comparatively and the

distribution of the values of the wound size was not normally distributed. Hence,

nonparametric statistical test of significance – Wilcoxon’s Signed Rank test for within

group comparison and Wilcoxon’s Rank Sum test for between groups comparisons

were applied for statistical significance tests. In order to assess the time-to-complete

healing and the probability of wound healing, Kaplan- Meier curves were constructed

and compared between the arms with the use of Log-rank test, indicating the

superiority of fluconazole therapy. All reported “p” values are two-tailed. Analyses

were performed using SPSS software, version 17.0.

Results

From January 2008 through June 2010, a total of 518 patients hospitalized for surgical

management of DLWs were screened. 75 of 111 eligible patients volunteered to

participate in the study. 38 patients were given fluconazole plus standard care; and 37

patients were given only standard care.

The base-line characteristics of each of the study arms are shown in Table 1. The

mean age, sex, duration of diabetes, TcPO2, ABI, VPT, HbA1c, ALT, AST and

serum creatinine were comparable between the two study groups. No significant

differences in liver and renal functions were observed between the arms at the end of

the study. Both the groups had severe diabetic peripheral neuropathy, uncontrolled

HbA1c and good tissue oxygen perfusion.

Table 4.1. Baseline Characteristics of the patients.

DM- diabetes mellitus, TcPO2- transcutaneous partial pressure of oxygen, ABI-

Ankle brachial index, VPT- vibration perception threshold, HbA1c- glycosylated

hemoglobin A1c, AST- aspartate aminotransaminase, ALT- alanine aminotransferase,

SA- surface area

The surgeries performed between two arms were comparable (table 4.1). About 45%

(34/75) of the population had undergone major debridement (wound surface area

>10cm2

after debridement), 22.6% (17/75) underwent minor debridement (wound

surface area </= 10cm2

after debridement) and 13.3% (10/75) had mid-foot

amputation done. All patients had an open wound post operatively. The commonest

fungi isolated were C. parapsilosis, C. tropicalis, C.albicans etc and the predominant

bacterial isolates were Enterococuss faecalis, E.coli and Staphylococcus aureus,

which were comparable between the two groups (table 2).

Table 4.2. Fungi and bacteria isolated from deep tissue of Diabetic Lower

Extremity Wounds.

As shown in figure 4.5, at baseline, the mean wound SA was 111.51cm2 in the test

arm. Whereas, it was to 87.25cm2 in the control arm. By week 4, the SA reduced to

25.9cm2 in the test and to 67.06cm

2 in the control arm. Thereafter, significant

decreasing trend of wound SA was observed in the test arm compared to the control

arm. Statistically significant (p</=0.05) difference in mean WSA was observed at

week 6 between the T and C.

Figure 4.5. Trend of wound reduction between test and control arms over 14

weeks.

Twenty patients in T and 24 patients in C had complete healing, but this difference

was not statistically significant (p=0.47). The estimated mean time for complete

wound healing was 7.3 weeks for the T and 11.3 weeks for the C at 95% confidence

interval. The estimated median time for complete wound healing was 6 and 10 weeks

for the treatment and control group respectively. This difference was statistically

significant (p=0.022) as shown in figure 4.6. Figure 4.7 shows the probability of

patients estimated to have complete wound healing with and without fluconazole

therapy for deep tissue fungal infections in DLWs. It is found that by week 4, 50% for

patients in treatment group would have complete wound healing , as against 20% in

the control group.

Figure 4.6. Kaplan- Meier method showing the mean and median time for

complete wound healing between the test and control arm.

Figure 4.7. Kaplan- Meier method showing the probability of complete wound

healing among the patients with and without fluconazole therapy for deep tissue

fungal infections in DLWs.

Four patients in T arm and one patient in C arm were not offloading their foot during

the study; hence were not included for final analysis. 2 patients in the T and one

patient in C expired due to cardiac cause. Twelve patients in T and eleven in C

stopped coming to our podiatry clinic for follow-up visits during due to social and

financial problems.

Table.4.3. Details of wound site, duration, wound surface area at entry and end

of the study; and time to complete wound healing of the study subjects.

SID1 Wound Site WD2

Initial

WSA3

WSA

(EOS4)

TTC

H SID1 Wound Site WD2

Initial

WSA3

WSA

(EOS4)

TTC

H

1 Fore-foot plantar area 23 58 LTF NA 38 Hind-foot plantar area 21 42.25

24.75(4

) NA

2 Fore-foot plantar area 15 37.11 4(18) NA 39 Hind-foot plantar area 20 41.25 LTF NA

3 Mid-foot dorsum 28 31.06 0.5(10) NA 40

2nd Toe & Fore-foot plantar

area 21 60 LTF NA

4 Foot plantar area 24 95 18(12) NA 41 Sole of foot 28 123.5 178(2) NA

5 Foot plantar area 30 104 LTF NA 42 Hind-foot plantar area 10 26.6 15.6(4) NA

6 Mid-foot plantar area 9 4.8 0 8 43 Foot dorsum & plantar area 21 287.5 Expired NA

7

Fore-foot & mid-foot

dorsum 37 120 0 16 44 Sole of foot 30 100 7.5(10) NA

8 Fore-foot plantar area 30 84.4 72(4) 4 45 Foot plantar area 14 20 LTF NA

9 Fore-foot plantar area 14 32.4 0 10 46 Foot plantar area 15 42.4 30(4) NA

10 Mid-foot plantar area 19 42 0 16 47 Sole of foot 30 97.2 0 2

11

Fore-foot & mid-foot

dorsum 21 178.5 0 16 48

1st Toe and Fore-foot plantar

area 28 72 0 2

12 Fore-foot dorsum 18 44 LTF NA 49 Mid-foot dorsum 17 34.4 0 4

13 2nd Toe & Sole-Foot 18 80.6 0 2 50 Foot plantar area 30 152.95 0 6

14 Mid-foot plantar area 13 72 0 10 51 Fore-foot plantar area 14 6.9 0 6

15 Fore-foot plantar area 12 20 7.5(12) NA 52 Fore-foot plantar area 20 75 0 8

16 Fore-foot plantar area 24 41.25 20.5(8) NA 53 5th Toe 14 7 2.4(6) NA

17 Fore-foot plantar area 18 8.62 0 12 54 Fore-foot plantar area 6 4.1 0 4

18 Mid-foot dorsum 18 23.65 57(12) NA 55 Fore-foot plantar area 16 33 0 10

19 Foot plantar area 21 82.66 0 10 56 Fore-foot plantar area 8 9.6 0 12

20 1st Toe & Dorsum-foot 30 175.6 LTF NA 57 Fore-foot plantar area 12 9 0 14

21 Fore-foot plantar area 14 4.42 6(2) NA 58 Mid-foot dorsum 7 1.29 0 4

22 Fore-foot plantar area 30 108 6(12) NA 59 Hind-foot plantar area 24 44.1 0 4

23 Fore-foot plantar area 42 151.75

21.28(24

) NA 60 Foot dorsum & plantar area 42 370.5 0 6

24 Fore-foot plantar area 12 10.4 13(22) NA 61 Sole of foot 21 164 0 14

25 Fore-foot plantar area 12 7 0 4 62 Foot dorsum & plantar area 35 584.24 Expired NA

26 Fore-foot plantar area 21 31.5 0 4 63 Foot dorsum & plantar area 30 546 559(2) NA

27 Fore-foot plantar area 16 12.6 0 6 64 Fore-foot plantar area 14 16

0.75(12

) NA

28 Fore-foot plantar area 7 5.16 0 2 65 Foot dorsum & plantar area 38 528 0 2

29 Fore-foot plantar area 24 47.5 0 10 66 Fore-foot plantar area 30 62.5 0 22

30 Fore-foot plantar area 7 4.25 0 4 67 Foot dorsum & plantar area 38 257.5 272(4) NA

31 Mid-foot dorsum 14 13 LTF NA 68 Foot dorsum & plantar area 30 270 0 2

32 Mid-foot dorsum 20 84 0 8 69 Foot dorsum & plantar area 46 894 Expired NA

33 Fore-foot plantar area 16 5.95 0 4 70 Mid-foot plantar area 7 4.8 0 2

34 Mid-foot dorsum 24 96 LTF NA 71 Mid-foot dorsum 14 13.65 0 12

35 Mid-foot dorsum 15 21 0 10 72 Fore-foot plantar area 12 37 0 10

36

Foot dorsum & plantar

area 21 337.5 0 12 73 Fore-foot plantar area 21 69.75 0 12

37 Fore-foot plantar area 27 50.35 0 32 74 Fore-foot plantar area 25 54 0 12

75 Fore-foot plantar area 30 42.55 0 12

1.Subject Identification number,.2. Duration of Wound in Days, 3. Wound Surface Area (in

Centimeter Square), 4. End of Study (in weeks), 5. Time to Complete Healing (in weeks),

LTF = Lost to Follow Up, NA= Not Applicable.

Discussion

For the first time, our study demonstrates that antifungal therapy for deep seated

fungal infections in DLWs hasten not only wound reduction but also complete

healing. We observed a rapid decrease in mean wound size in T compared to C in our

study. Significant wound reduction was observed as early as 4 weeks among the

treatment group compared to the control group. The chance of complete wound

healing at week 4 was found to be about 50% in treatment group as against about 20%

in control group in Kaplan-Meier survival analysis (figure 2B). Percentage change in

foot ulcer area after 4 weeks was found to be a robust predictor of healing (130).

Sheehan et al demonstrated that, absolute change in wound area at 4 weeks was

significantly greater in healers versus non-healers (130). It was reported that open

wounds with larger surface area cause heat loss and hypothermia, which in turn cause

tissue hypoxia, impaired cellular immune functions, increased risk for infections and

delayed wound healing (45). Rapid reduction in wound size observed in our treatment

arm would have prevented desiccation of wounds and enhanced healing. Our study

demonstrates the superiority of antifungal therapy for deep seated fungal infections in

wounds over standard care. On analysis of our data, the mean time for complete

wound healing was found to be 7.3 weeks in test arm compared to 11.3 weeks in

control arm. In contrast, it was 15 weeks in Moretti et.al’s and 18.5 weeks in Jeffcoate

et.al’s intention to treat randomized control trials (66, 101). The possibility of deep

seated fungal infections was not carefully looked at in the previous studies.

About 96% of all fungal species and majority of the Candida are susceptible to

fluconazole (32). It is also one of the cost effective, commonly available and potent

fungistatic agents that can be administered orally at dose between 3-12mg/kg/day for

invasive candidiasis (73). Pharmacological properties of fluconazole offer many

advantages including its metabolism, bioavailability, renal elimination of active drug,

penetration of tissue and cerebro-spinal fluid, and safety profile (26, 50, 107, 157).

Our study patients had good tissue oxygen perfusion around the wounds, which

certainly would have allowed the drug to reach deeper into the DLWs and inhibit

fungal growth. Continuous or intermittent antifungal therapy (fluconazole, terbinafine

and itraconazole) had been found effective against dermatophytosis and

onychomycosis, but the effect of these regimens for treating deep seated fungal

infections in DLWs have not been studied so far. Studies recommend greater doses of

fluconazole administration to maintain higher therapeutic concentration especially

while treating invasive fungal infections (73). Therefore, though plasma half life of

fluconazole is 30 hours, we gave fixed dose of 150mg fluconazole daily to T arm who

had significant wound reduction even at week 4 compared to C arm. The role of

intermittent or pulse antifungal therapy for deep tissue fungal infections in DLWs is

yet to be explored. Studies showed a low incidence of adverse events related with

fluconazole (31). Three patients in T arm had reported mild gastrointestinal symptoms

which did not prevent our patients from continuing in the study. Liver and renal

functions of our patients were within normal limits at the end of the study.

Though, total number of subjects who met primary outcome was more in C arm

compared to T arm, but the difference was not statistically significant (p=0.47).

Overall patient compliance to the treatment was good in T as well as C arm. In figure

1, the trend of wound reduction at follow-up visits was observed to be smooth except

at week 12 in T arm; and at week 10 and 14 in C arm. It may be because that by week

10, majority of our patients had complete wound healing. And, by the end of the

study, only a small number of patients remained, whose mean wound SA may have

distorted (at week 12) the smooth down slop of the trend graph (shown in figure 1).

Foot offloading and regular follow-up visits till the time of wound healing has been a

challenge in any wound studies. Improper offloading could be another contributory

factor for the increased wound SA at week 10, 12 and 14 among the subjects.

There were a few short comings in our study. Firstly, patients with a WSA >/=4cm2

were included in the study. We did not keep an upper limit for wound surface area.

Though, upper limit for WSA would have slowed down the patient recruited, it would

have maintained the normal distribution of the WSA for both the groups. Secondly,

there were >20% drop-outs or lost to follow-ups in each arm. This might include

baseline wound size comparison, renal and liver functions for those that completed

the study versus those that were lost. And not all patients were visiting our podiatry

clinic every 2 week for follow-up assessment. Therefore, a bigger sample size would

have taken care of this deficiency. Thirdly, in order to avoid trauma and iatrogenic

ulceration, we did not perform post-treatment fungal cultures to evaluate mycological

cure in the treatment group.

Multiple risk factors involve in healing of DLWs and hence each risk factor is a target

for clinical intervention. Studies have shown that management of diabetic foot ulcers

is based on 3 principles; firstly, maintaining strict glycemic control; secondly,

thorough debridement of necrotic and inflamed tissue in the wound, regular

inspection, cleansing , elimination of pathogens and creating of an appropriate

environment to facilitate endogenous tissue regeneration. Lastly, on promoting wound

healing by surgical revascularization, dressings, applying technologies which would

stimulate the release of growth factors, cytokines (65).

To conclude, Fluconazole plus standard care was superior to standard care alone in

accelerating wound reduction and complete wound healing among diabetic patients

with deep seated fungal infections in their lower extremity. Therefore, every diabetic

patient with DLWs has to be screened for fungi as well as bacteria; and treated with

culture specific antifungal and antibacterial agents. Further studies are warranted on

formulating different treatment regimen and to learn the effect of other antifungal

agents in DLW management.