FUNGAL ETIOLOGY OF DIABETIC

FOOT ULCER
Dr. ASHOKAN. K. KUTTIYIL Date:10.10.10
Department of Microbiology Place: Calicut
Medical College, Calicut-673008

CERTIFICATE

This is to certify that the project work entitled “FUNGAL ETIOLOGY OF

DIABETIC FOOT ULCER” to be submitted to The Department of Medical
Microbiology, School of Health Sciences, Kannur University, Thalassery
Campus, Thalassery in partial fulfillment of the degree of Master of Science in
Medical Microbiology is a bonafide record of original dissertation work carried
out by AFSEENA.M.P. under my guidance and supervision at the Department
of Microbiology, Diabetes Hospital, Calicut, during July to September 2010
and the project work has not formed the basis of any Degree/ Diploma/
Associateship/ Fellowship or other similar to any candidate in any University.

Dr: ASHOKAN. K. KUTTIYIL

(Guide)
‘
 DECLARATION

I, AFSEENA.M.P. hereby declare that the project work entitled “FUNGAL

ETIOLOGY OF DIABETIC FOOT ULCER” submitted to The Department of
Medical Microbiology, School of Health Sciences, Kannur University,
Thalassery Campus, Thalassery in partial fulfillment of the degree of Master
of Science in Medical Microbiology is a record of original diissertation carried
out by me under the guidance of Dr. ASHOKAN. K. KUTTIYIL, Senior
Scientific Assistant, Department of Microbiology, Medical College, Calicut. It
has not formed the basis of any Degree/ Associate ship/ Fellowship or any
other similar title to any other candidate to any University.

AFSEENA.M.P.
 ACKNOWLEDGEMENT

First of all, I would like to thank my supervisor Dr. Ashokan.K.Kuttiyil, for

his excellent guidance, understanding, encouragement and help throughout
this study.

Also I would like to express my grateful thanks to Mr.Kasim. M. (Lab in

Charge) Mr. Rejadeesh (Microbiologist), Mr. Fazal and all staff members of
Diabetes Hospital Hospital, Calicut, for their constant support and valuable
comments during thesis study.

Special thanks are extended to Dr. Moideen, Diabetes Hospital, Calicut

and Dr. Ramamoorthy,Malabar Diabetes foundation and research centre, for
his valuable Suggestions, comments and kind helps.

I am thankful to Dr. Sangeetha, Dr. Shiraz Abdul Rauf, Dr. Deepthi, Dr.
Ummer, Chest and Diabetes Hospital, Calicut, for their timely help and
support.

I express my sincere thanks and heartfelt gratitude to Mr.Abdulla,

Managing Director, Diabetes Hospital, and Calicut

I also express my special thanks to all staff of Ideal Graphics, Calicut ,

Global Printers, Payyoli and Net Camp, Vadakara.

It is not only my pleasure but also my duty to thank all the staff
members of School of Health Sciences, Kannur University, Thalassery
Campus, and Thalassery for their kind support and encouragement during my
work.

Finally, I want to express my gratitude to my friends and family. Thanks

for all their motivation, encouragement and their support throughout not only
this study and also all through my life.

Place: Calicut
Date: 10.10.10
AFSEENA .M.P.
 INTRODUCTION AND REVIEW OF LITERATURE

Diabetes mellitus has been recognized as a medical condition since the first century
AD. It is estimated today that diabetes mellitus affect 15% of all people in developed
countries world wide. The morbidity & mortality of diabetes mellitus is staggering. Diabetes
is the leading cause of blindness, kidney disease and non traumatic limb loss in the world.

The diabetic foot is considered one of the most significant complications of

diabetes representing a major world wide medical, social and economic problem that
greatly affects patient quality of life. The risk of patient with diabetes to develop a foot
ulcer is close to 25% (Sing et al, 2005) leading frequently to disablement and leg
amputation (Precoraro et al, 1990). It has been estimated that every 30 seconds a lower
limb is amputed somewhere in the world because of diabetes (International Diabetes
Federation, 2005). The link between the diabetic foot ulcer and leg amputation is
indisputable, as diabetes is the cause of almost 50% of all non traumatic lower extremity
amputations world wide. Early diagnosis and efficient treatment of the diabetic foot
ulceration is essential in order to avoid limb amputation and preserve the life quality of
patient with diabetics. The alarming fact is that India has more people with diabetes than
any other country (Wild et al, 2000) and the incidence of foot problems and amputation
remains very high because of several practices such as barefoot walking , inadequate
facilities of diabetic care, low socio economic status and illiteracy. Foot infections among
diabetic patients are common ranging from chronic bacterial or fungal infections to
serious limb threatening ones. The present the study is focused on fungal agents that
causing diabetic foot ulcer.

The word ‘mycology’ in fact, is derived from ‘mykes’, the Greek word for mushroom.
Medical mycology has emerged as an important branch of Microbiology due to increase in
the isolation of opportunistic fungal pathogens especially in immunocompromised patients.
Organisms once thought to be contaminants are not considered as pathogens in
compromised patients. Fungal infections, however, are extremely common and some of
them are very serious and even fatal.
 Diabetes mellitus, often simply referred to as diabetes is a condition in which a
person has high blood sugar, either because the body does not produce enough insulin
(Type 1) or because cells do not respond to insulin that is produced (Type 2). It has been
recognized as a medical condition since first century AD when Cappadocia coined the
condition term diabetes, meaning siphon.

Diabetes mellitus affect all socio economic age groups and the disease affects
approximately 60 million people worldwide, 16 million in USA and 1.5 million in Canada
(A.K.Gupta et al, 2000). However between 1958 and 1993 the number of individuals
diagnosed with Diabetes mellitus will increase fivefold. It has been estimated by the WHO
that the incidence will raise to 300 million by the year 2025.

SYMPTOMS
 Polyuria
 Polydypsia
 Polyphagia
 Blurred vision
 Weight loss
 Dry, itchy skin
 Loss the feeling their feet
 Having sores that heal slowly
Diabetic patients may present with complications involving all systems of the body,
including:
 Neuropathy & impaired circulation
 Renal disease
 Cardio Vascular disease
 Retinopathy
 The development of several skin manifestations.
 Diabetes mellitus is characterized by recurrent or persistent hyperglycemia, and is
diagnosed by demonstration anyone of the following
 Fasting plasma glucose level > 126 mg/dl
 Plasma glucose > 200 mg / dl, 2 hrs after a 75g oral glucose load

2006 WHO Diabetes Criteria

Condition 2 hour glucose fasting glucose

(mg/dl) (mg/dl)

Normal < 140 <110

DM > 200 > 126

 Symptoms of hyperglycemia and causal plasma glucose > 200 mg/dl

 Glycated hemoglobin Hb A1C > 6.5%
 Diabetic Foot & Diabetic Foot Ulcer

Diabetic foot is an umbrella term for foot problems in patients with diabetes
mellitus. Due to arterial abnormalities and diabetic neuropathy, as well as a tendency to
delayed wound healing, infection of foot is relatively common. 10-15% of diabetes patient
develops foot ulcers at some point in their lives and foot related problems are responsible
for up to 50% diabetes related hospital admission.

Diabetic foot ulcer is one of the major complications of diabetes mellitus. It occurs
in 15% of all patients with diabetes and proceeds 84% of all lower leg amputations.
(Harold Brem et al, 2001) Major increase in mortality among diabetic patients, observed
over the past 20 years is considered to be due to the development of macro and
micro vascular complications, including failure of the wound healing process.
‘Wound healing’ is a ‘make-up’ phenomenon for the portion of tissue that gets
destroyed in any open or closed injury to the skin. Being a natural phenomenon,
wound healing is usually taken care of by the body’s innate mechanism of action
that works reliably most of the time. Skin serves as barriers between the internal
organs and the external environment. The skin covering the body, protect it considerably
against invasion by microorganism

From inside out the skin is divided in to 3 distinct layers:

 the subcutaneous tissue
 the dermis
 the epidermis

Subcutaneous tissue lies beneath the dermis and is rich in fat. Deep hair
follicles and sweat glands originate in this layer. Below subcutaneous layer are thin
facial membranes (sheets or bands of fibrous tissues) that cover muscles, ligaments
and other connective tissues of importance, the fascia serves as a barrier of infection for
the deeper tissue and organs of the body. Above subcutaneous tissue and facial
membranes lies the dermis, comprises dense connective tissue that is rich in blood and
nerve supply. Shorter hair follicles and sebaceous glands originate in dermis. Finally
epidermis, which is the outer most layer of skin, is made of layered squamous
epithelium. Hair follicles sebaceous glands and sweat glands open to the skin surface
through the epidermis

Key features of wound healing are stepwise repair of foot extracellular matrix that
forms largest component of dermal skin layer (Nomikos et al, 2006). Therefore controlled
and accurate rebuilding becomes essential to avoid under or over healing that may
lead to various abnormalities. But is some cases, certain disorders or physiological insult
disturbs wound healing process that otherwise goes very smoothly in an orderly
manner. Diabetic mellitus is one such metabolic disorders that impedes normal steps
of wound healing which include

 Haemostatic or inflammatory phase in which damaged tissue releases

chemical mediators called cytokines (TGFβ). Platelets aggregate to stem
bleeding and releases serotonin and other vasoconstrictors and activate
coagulation cascade. Monocyte and neutrophil are attached the site of injury.
Neutrophil trap and kill micro organism immediately while monocytes become
activated macrophage which produce growth factors and cytokines and
scavenge nonviable tissue and various microbes. Angiogenic growth factors
stimulate neovascularisation of wound bed.

 In proliferative phase macrophage recruit fibroblast which creates a network of

collagen fibers. When adequate O 2 and vitamin C are present, granulation
tissue forms. O 2 is incorporated by 2 amino acids proline and lysines which are
both required for collagen synthesis. vitamin C is required for the hydroxylation of
proline to hydroxy proline an amino acid found in collagen.
  Remodeling phase in which an organized form of collagen gradually replaces
the immature soft, gelatinous collagen. The effect is to increase the tensile
strength of the healed wound.

Diabetes Mellitus is a metabolic disorder and hence the defects observed in

diabetic wound healing are thought to be result of altered protein and- lipid metabolism
and thereby abnormal granulation tissue formation (Janet Close et al, 2002) which
result in the formation of advanced glycation end products (AGES) or Amadori
products which alter the properties of matrix proteins (Collagen, vitronectin and laminin)
(Alison Goldin et al, 2006) (Singh R et al, 2001).

Another important factor contribute to poor wound healing is impaired Nitric oxide
synthesis which increases fibroblast proliferation and thereby collagen production (Kei
obayashi et al, 2006) (Dan G Duda et al, 2004). It may be due to the accumulation of
Nitric Oxide synthase inhibitor due to high glucose level in diabetes mellitus.
Fibroblast from diabetic ulcer exhibit proliferative impairment that probably contributes
to decreased production of extracellular matrix proteins and delayed wound healing
(Miriam et al, 1999).In diabetes their occur a reduced levels of TGFβ), lowers down the
inhibitory regulatory effect on matrix metalloproteinases(MMP)genes and thus cause
matrix metalloproteinase to over express (Neil Burnett et al, 1993) ( Galkowska et
al, 2006) which degrade almost all the extra cellular matrix components.
 Pathophysiology of Ulceration

A person with diabetes may not be able to feel his or her feel properly due to
damage to nervous systems. Normal sweat secretion and oil production that lubricates
the skin, bones, and joints of the foot is impaired. These factors together can lead to
abnormal pressure on the skin, bones and joints of foot during walking and can lead to
break down of skin of foot. Damage to the blood vessels and impairment of immune
system from diabetes makes it difficult to heal these wounds. Microbial infection of skin,
connective tissues, muscles and bones can then occur. These infections can develop
in to gangrene or ulcers. Because of the poor blood flow, antibiotics cannot get to the
site of the infection easily; the only treatment for this is amputation of foot or leg. If the
infection spread to the bloodstreams, this process can be life threatening.

1. Neuropathy (sensory, motor and autonomic) is most important cause (Vlbrecht et al

2004)

Sensory
 Loss of pain
 Pressure awareness
 temperature
 Proprioception
Motor
 Atrophy
 Intrinsic muscle weakness
(Toe deformity and abnormal walking pattern)
 Increased pressure areas
Autonomic - Reduction or absence of sweating causes dry cracks, fissures in
the skin susceptible to fungal and other superficial infection

2. Vascular Insufficiency

 Peripheral Vascular Disease (PVD) with minor or trival injury leads to painful
ischemic lesion

3. Infection
 Diabetic patients became more susceptible to bacterial fungal and yeast infection due
to medical and nutritional changes that take place in body. Most of the diabetic foot
infections are polymicrobial in nature and mixed organisms are frequently
encountered. The spectrum of micro depends mainly or microbial flora of the lower
limb, metabolic factors, foot hygiene and the use of antibiotics. Emergence of
resistance among organisms against the commonly used antibiotics has been clearly
outlined in various studies as being largely due to their indiscriminate use. In diabetic
patients, mycotic infections may increase the risk of developing diabetic foot
syndrome Candida Sp is the most commonly isolated yeast from ulcers (5%-21%).
Environmental fungi including Aspergillus, Alternaria and Fusarium can produce
infection and toxin related disease.

Given the condition prevailing in diabetic foot, even low pathogenic yeast may cause
infection of foot ulcers. These types of yeast often belong to normal microbiota of the
skin around ulcers or may colonize diabetic foot ulcer, secondarily hindering the
assessment of the real role of fungal isolates from the ulcer.

4. Biomechanics
 Development of foot ulcer in Diabetic foot

DIABETES MELLITUS

NEUROPATHY PERIPHERAL MICRO

VASCULAR VASCULAR ANGIOPATHY
DISEASE
(PVD)
SOMATIC AUTONOMIC

Reduce pain Restricted Absence of

and Impaired blood FOOT ISCHEMIA
joint mobility pressure flow regulation
proprioception

Dry Skin Dilated foot

INCREASES D tissues veins, dry foot
FOOT
PRESSURE

Small muscle CALLUS

weakness

FOOT ULCERATION INFECTION

AMPUTATION
‘

Classification of diabetic foot ulcer

The evaluation and classification of diabetic foot ulcer are essential in order to
organize the appropriate treatment plan and follow up. During the past years several foot
ulcer classification methods have been proposed, however none of the proposals have
been universally accepted.
Stage A - Clean wounds
Stage B - Non ischemic infected wounds
Stage C - ischemic wounds
Stage D - infected ischemic wounds

In a simplified clinical classification approach, diabetic foot ulcers can be

characterized. The Wagner- Meggit classification (Oyibo et al, 2001) is based a mainly
on wound depth and consist of 6 wound grades. Theses include:

Grade 0 - Intact skin

Grade 1 - Superficial ulcer
Grade 2 - Deep ulcer to tendon, bone or joint
Grade 3 - Deep ulcers with abscess or osteomyelities
Grade 4 - Fore foot gangrene
Grade 5 - Whole foot gangrene

The university of Texas system grades the ulcers by depth and then stages them
by the presence or absence of infection and ischemia.
i.e., Grade 0 - Pre or post ulcerative site
 Grade 1 - Superficial wounds through either the
epidermis or the epidermis and dermis but that
do not penetrate to tendon, capsule or bone.
Grade 2 - penetrate to tendon or capsule but bone and
joints are not involved
Grade3 - Penetrate to bone or in to a joint

Fungi Causing Diabetic Foot Ulcer

Fungal skin infection is common in patient with diabetes ( Missoni et al 2006),

and it is the most probable fact of delayed wound healing. Fungi seldom behave as a
pathogen in normal host but occur most often in immunocompromised host with or
without underlying pathology. Fungi are initially classified with the plants and much of the
botanical influence is still seen, even though the organisms have been transferred to a
separate kingdom on the basis of cell structure. Fungi are ubiquitous nature. They are
eukaryotic and cell wall containing chitin and/ or cellulose, chemo-heterotrophic. They
function as saprophytes and also as a decomposer in nature. Of the estimated 25,000
species, less than 150 are known to be primary pathogens of humans.

Fungi reproduce by the formation of spores, which may be either asexual (involving
mitosis only) or sexual (involving meiosis; preceded by the fusion of the protoplasm and
nuclei of two cells). Specialized structures (fruiting bodies) may be associated with either
sexual or asexual spores and are helpful for identification. Asexual spores are of two
types: sporangiospores and conidia. Sporangiospores are characteristic of lower fungi,
zygomycetes. Conidia are asexual spores of higher fungi. They are represented by the
classes Ascomycetes, Basidiomycetes, and Deuteromycetes. The sexual spores of
Ascomycetes is the ascospore, basidiomycetes is the basidiospore. The Deuteromycetes
(Fungi imperfecti) have no sexual spores.

Diabetic patients are at an high risk of infections caused by opportunistic fungi

such as yeasts [Candida, Cryptococcus] and the dust fungus). Environmental fungi
include Aspergillus, Alternaria and Fusarium are known to produce toxin related
diseases. The presence of various fungal pathogens in diabetic foot-ulcer tissue was
shown by Chincholikar and Pal in 2002 among which Candida Sp preponderated.

Candida Sp are frequently the primary pathogen followed by bacterial infection

with streptococcus, staphylococci, pseudomonas and coliforms (MC Carty et al, 1994).The
presence of various species of Candida ( C albicans, C tropicals, C parapsilosis, C
guillermondi, C krusei ) was reported in diabetic patient with ulcer (Missoni. et a1, 2005),
Chakrabarthi et al , 1996 reported C tropicalis as the predominate isolate. c albicans in a
necrotizing soft tissue infection that developed around an operation incision scar was
described in a renal transplant patient with diabetes mellitus ( wai et al, 2000).
Fournier gangrene due to Candida Sp was also reported in some patient's (Jhonin et al,
2008) (Loulergue et al, 2008). A case of untreated diabetes mellitus and an ulcer on the
perineum resulting in necrotizing soft tissue infection with candidemia by c glabrata was
reported by (Shinto et al, 2009). Another case of isolated c ablicans skin abcess in a
critically ill patient with a history of intraabdominal surgery and candidemia was reported
by (Tuon et al, 2006)

Molds were also isolated from diabetic foot ulcer of which Aspergillus sp
predominated. The other mold isolated were Fusarium sp, penicillum marneffi and
basidiobolus ranarum

The presence of A flavus in diabetic foot-ulcer has been first reported by ( Bade et
al, 2003).) A case of simultaneous aspergillosis and mucoromycosis complicating
diabetic foot gangrene was reported by (Reyes et al,1984). P marneffei has been rarely
reported in India ( Forbes et al, 2002)The study conducted by Seema Nair et al, 2006
signifies the need of mycological evaluation of non healing diabetic foot and the incidence
of mycotic infection in diabetic foot tissue. They had studied 74 ulcer cases over a
period of one year in Kochi in which 65% had yeast and mold infection.

Fusarium sp, a mold which causes disease mainly in plants has emerged as
a pathogen in immunocompromised patient especially in those under long term steroid
therapy. Main route of acquisition is through direct inoculation (Hospenthal et al, 2005)
(Magnini et al, 1999) .The fungal culture of escher of ulcer from a patient who was
under antibacterial drug for two months, still the ulcer did not heal isolated
Fusarium solani (Ramakrishna Pai et al, 2010) , this case gives emphasis on fungal
culture in chronic diabetic ulcer. The most commonly isolated Fusarium from clinical
specimen is Fusarium solani followed by Fusarium oxysporum and Fusarium monilformis
(Campell et al, 1996), ( Bader et al, 2003). A conventional and molecular study isolated
Fusarium sporotrichoides from the patient's diabetic foot with a history of enjoying
walking barefoot (Mustafa Ozyurt et al, 2008). T2 toxin production of the pathogen was
investigated using HPLC (Jimenez et al, 1997).

A study conducted by, Acta Med Croatica, 2006 in 509 diabetic patient cases in
33.85% out of foot ulcer patients, the infection were confirmed by a finding of fungal
elements in histopathologic preparation of ulcer biopsy specimens. Fifteen species from
the genera : Candida, Cryptococus, Trichosporon and Rhodotorula were the causative
agents.

The pathogenic effect of yeast in foot ulcer is indicated by the severity of clinical
findings, chronic course of infection and infection progression despite antibiotic therapy.
There have been some reports of an increased incidence of fungal infection such as
dermatophytosis and candidiasis of interdigital spaces and nails in the toes of diabetic
patients as well as of the association of these infections with the development of severe
and deep inflammatory process in feet (Rich et al, 1999) ( Gupta et al, 2000).
The studies on foot ulcer (Emilija Milnaric Missoni et al, 2005) showed that
coexistent interdigital colonization with yeast and dermatophytosis has no impact in the
incidence of fungal diabetic foot ulcer infection.

Absida corymbifera and Saksena Vasiformis have been reported in cutaneous

zygomycosis particularly among diabetic patients (Jagdish Chander, 1995). In diabetic
patients mycotic infections may increase the risk of developing diabetic foot syndrome.
 Pathogenesis and Clinical features

A diabetic foot infection is most simply defined as any inframelleolar infection in a

person with diabetes mellitus. These include paronychia, cellulutis, abcesses,
myositis, necrotizing fascitis, septic arthritis, tendonitis and osteomyelitis. The most
common and classical leision, however is the infected diabetic `mal perforans', foot
ulcer.

Various immunologic disturbances, especially those involve polymorphonuclear

leukocytes, may affect some diabetic patients, that likely increase the risk and severity of
foot infection (Schubert et al, 1995), (Gin et al,1993),( Joshi et al, 1999), (Geerlings et al),
1999)
The risk of infection from fungi are increase because of decreased cellular
immunity caused by acute hyperglycemia and circulatory defects caused by
chronic hyperglycemia (Lipskey et al 1999)( Mandelm et al 1978) .The large size of
fungi protect them from being phagocytosed (MEEI immunology service 1999.
Polymorphonuclear leukocytes known to be pivotal in penetrating fungal infection since
they phagocytose and subsequently destroy fungal structure by oxygen depend
mechanism. The use of steroids may interfere with this mechanism and hence lower the
host-resistance to fungal infection. Also ketoacidosis and altered white cell chemotactic
ability that may render diabetes patient susceptible to fungal infections (Mandel et
al,1978). Such fungal infections may result in cracks in the toe clefts and lead to
secondary bacterial infections (Lipsky et al 1999). In addition, repetitive insults to
thickened soles or callosities on the feet may lead to deep fissures and cracks that open
the door to infection with fungi.

This wound results from a complex amalgam of risk factors (Caputo et al, 1994)
(Frykber etal, 1998) which include peripheral neuropathy (motor, sensory, autonomic)
Neuro-osteo arthropathic deformities (Charcot disease),or limited joint mobility,
vascular insufficiency, Hyperglycemia and other metabolic derangements impaired
immunological. especially neutrophil function and wound healing and excess collagen
crosslinking, patient disabilities (reduced vision, limited mobility and previous
amputation), Maladaptive patient behaviors (inadequate precautionary measures and
foot inspection and hygiene procedures poor compliance with medical care,
inappropriate activities, excessive weight bearing and poor foot-wear), Health care
system failures inadequate patient education and monitoring of glycemia control and foot
care.

Once the protective layer of skin is breached, under lying tissues are exposed
to microbial colonisation. This wound may progress to become active infected due to
various virulence substances (adhesins, toxins etc) by the organism and by contiguous
extension, the infection can involve deeper tissue. This sequence of events can be rapid.
In case of Candida there occur a wide range of virulence factors which include Host
recognition biomolecules (adhesins) that helps the fungus to recognize and bind to
host cells, morphogenesis (the reversible transition between unicellular yeast and
filamentous growth form), secreted aspartyl proteases, phospholipases, phenotypic
switching accompanied by changes in antigen expression, colony morphology and
tissue affinities. Many species of Aspergillus produce enzymes and toxic metabolites that
inhibit macrophage and neutrophil phagocytosis and underlying immunosuppresion
affecting neutrophil number and function.

Symptoms
 Early symptoms of diabetic foot ulcer include redness of skin, blistering and other
signs of irritability. Later stages, the person may have an open wound that drains fluid. The
open wound then become infected and develops the following symptoms.

 Swelling of feet due to inflammation or infection

 Hair loss from lower legs and feet.
 Hard shiny skin on the legs, (poor circulation). Localized warmth can be an
important sign of infection perhaps from wounds that won't
 Wounds that heal or slow to heal
 Calluses and corns may be sign of chronic trauma
 Drainage of pus form a wound is a late sign of infection.
 A limp difficulty walking can be sign of joint problem serious infection
 New or lasting numbness may be also develop in later stages which may be sign
of nerve damage and increase the risk for leg and foot problems

Diabetic patients, in particular are at the high risk of developing serious

complications in lower extremities that can lead to amputation. If a diabetic foot ulcer
isn't treated early and effectively, a person may experience:

 an infection in the ulcer itself

 septicemia, an infection of blood stream, which can be caused by fungi from the
ulcer
 loss of function and ability to perform activities if daily living
 amputation of the involved foot or leg
 death
 peripheral arterial occlusive disease and sensory and autonomic neuropathy
(Occur as major complication in diabetic foot )
 Evaluation and Treatment of Diabetic Foot Ulcer

Foot ulcer evaluation should include assessment of neurological status, vascular

status and evaluation of wound itself. Neurological status can be determined whether the
patient has pro tective sensation. Vascular assessment is important for eventual ulcer
healing. It include checking pedal pulses and also checking lower extremity arterial
pressure by doppler and recording pulse volume wave forms. Transcutaneous oxygen
measurements are often useful in determining whether a foot wound can heal.

Ulcer evaluation should include documentation of wound's location, size, shape,

depth base and border. Joint or bone. X-rays should be ordered an all deep or infected
wound. Signs of infection, such as presence of cellulitis, odour or purulent discharge
should be documented (Ingrid et al,2004)
Treatment

Foot ulcer in patient with diabetes should be treated for several reasons such as
reducing the risk of infection and amputation. The primary goal in the treatment of foot
ulcers is to obtain healing as soon as possible. The faster the healing there is less chance
of infection. Successful treatment of diabetic foot ulcer consists of addressing three basic
issues.
 Debridement
 Offloading
 Infection control
Debridement

It consist of removal of all necrotic tissue, peri wound callus and foreign bodies
down to viable tissue. Proper debridement is necessary to decrease the risk of infection
and reduce peri wound pressure. After debridement the wound should be irrigated with
saline or cleanses and a dressing should be applied.

Dressings should be prevent tissue desiccation, absorb excess fluid and protect the
wound from contamination. There are hundreds of dressing on market, including hydro
gels, foams, calcium alginates, absorbent polymers, growth factors and skin replacements

Offloading
By using
 Total contact casts (TCC)
 Cast walker
 Post operative shoes or wedge shoes

Infection control

Routine bacteriological methods along with mycological techniques are used to

determine the infection set up by the fungus. Treatment of infection caused by fungus could
done with antifungal agent such as Amphotericin B, Voriconzole or Caspofungin. Clinical
response was poor with Fluconazole,an azole derivative which interact with cytochrome p-
450 enzyme systems in fungal cells,resulting in impaired ergostrrol biosynthesis.
Caspofungin(Echinocandin),a fungicidal prevent cell wall synthesis by blocking β (1-3)D
glucan synthase enzyme which was preferred to Amphotericin B due to renal toxicity gave
a good clinical result in patients. (Ayyul et al, 2010). Some of them were resistant to
antifungal. The role of antifungal agent in wound management needs to be evaluated
further.
Non surgical treatment
By using
 Therapeutic foot ware
 Rocker bottom shoes
 Carvill splint
 Scotch TM cast
 Human recombinant platelet derived growth factors (Becaplermin)
 Hyperbaric oxygen therapy (HBOT)
 Nitric oxide, as a powerful vasodilator
 light therapy such as LLLT (low level laser therapy)
 Negative pressure wound therapy
Surgical Treatment

The removal of devitalized tissue to control infection and creation of an environment

favorable for healing, while maximizing the structural and physical integrity of foot, is
the central goal in surgical intervention in treating diabetic foot infections. Osteomyelitis
frequently result form direct extension to bone from a neuropathic ulcer and is more
effectively treated by surgical resection. Amputation for diabetic foot infections should
be limited to removing all necrotic devitalized tissue and bone while sparing as much
skin and tissue as possible. Arterial reconstructive surgery is an important adjacent to
therapy and may be required to heal an amputation.

Vital Precautions
 Never put pressure by prolonged walking or standing on the affected foot
 Put feet up while sitting to facilitate proper blood flow
 Keep ulcer covered by dressing to keep them clean and warm
 Never let dressing get wet as this can lead dirt and germs to the ulcer
 Followed standard wound management as suggested by physician
 Inspect feet daily, including, between toes
 Proper diet control for glucose levels to be maintained
 Don't wait to treat minor foot problems
  Do not self treatment corns, callus or other foot problems.

Diabetic Foot Care Products

Diabetic foot care products are specially designed to help to prevent

complications. It include: a diabetic foot cream which help in moisturizing. Healthy and
soft skin resist infection. Some times antifungal foot cream is called far to fend off fungi. It
can help relive symptoms like itching and burning, pumic stone may come on handy for
treating excessive formation of calluses, blisters or sores, magnifying glass and a
mirror to examine feet, diabetic socks which prevent moisture and build up of
microorganisms that cause infections, which are made of nylon, acrylic, cotton and elastic
fibers. Good fitting shoes put less pressure in areas that commonly cause foot problems.
 LABORATORY DIAGNOSIS

Even though an opinion can be made regarding etiology of diabetic Foot ulcer cases
based on the clinical picture to some extent, confirmation by laboratory methods is
essential as considerable variation in clinical Picture are likely to occur. A standard
protocol should be adopted to maximize the recovery of potential pathogens.

SPECIMEN COLLECTION :

Specimens (pus, wound exudates or tissue biopsy) for

microbiological studies were taken from ulcer region .Pus and exudates were collected
from margins and base of the ulcers using sterile swab stick. It is then transported in a
clean and sterile test tube. Tissue biopsies are also taken with a sterile blade in a wedge
shape including base and margin of the ulcer along with wound swabs from the same site.
Tissue samples provide more accurate culture results than superficial swab specimens.

 Cleanse and debride lesion before obtaining specimens for culture in case of open
wound, tissue specimens base by means of curettage of biopsy are taken
 Swabbing is done in the debrided wound base. Avoid swabbing underside ulcers or
wound drainage
 it must be rapidly transport to the laboratory.
 Needle aspiration may be useful for obtaining purulent collections .

MACROSCOPY :

Mainly looking for colour ,odour of the collected sample.

CULTURE OF THE MATERIAL :
 Direct streaking of the specimen on solid media will help to distinguish valid
growth from contamination . Sabouraud’s dextrose agar ( Sabdex or sabouraud agar)is
used for growth and maintenance of fungi , developed by Raymond Sabouraud (1864-
1938) ,a French mycologist it must be prepared with an antibiotic (streptomycin ,
gentamycin, chloramphenicol , cyclohexamide , actidione etc.); individually or in
combination to inhibit bacterial growth and make it more selective (Stevens et al,
1981 ).Several selective medias is also employed for isolation and presumptive
identification of yeast and filamentous fungi.

STAINING OF THE MATERIAL

Many authors have recommended Gram’s , Geimsa, Gomori’s methanamine silver
stains .The result of Gram stain have been advocated as a guide to the initiation of
treatment. Geimsa stain helps to determine the types of inflammatory reaction. The silver
stain identify the fungi well, staining the wall of the organism black and the background
light green.

WET MOUNT PREPARATON

Wet mount preparation using 10% KOH is found to be highly sensitive

technique to detect fungal elements. Especially when stained with Lacto phenol cotton
blue or Indian ink (Thomas et al, 1994) observed a significantly high level of sensitivity
over Gram stained smears for detection of fungal elements.
 AIM OF STUDY

 To identify the fungal agents causing diabetic foot ulcer in patients attending to a
Diabetes Hospital during a period of three months.

 Characterization and identification of different fungal isolates using routine

mycological technique

 Differentiation of various Candida sp using CHROM agar Candida.

 MATERIALS AND METHOD :

All the diabetic foot ulcer cases presented to the DIABETES HOSPITAL, CALICUT,
during the period July 2010 –September 2010 were studied. History of the disease
including

 Type of diabetes

 Duration of diabetes

 Size and depth of ulcer

 Any medication prior to hospital admission

 Any previous amputation

 Personal habits like smoking, alcohol consumption were recorded

SPECIMENCOLLECTION

Specimens such as pus, wound exudates or tissue biopsy were taken

from ulcer region. Cleansed and debrided the lesion before obtaining the specimen.
Tissue specimen is collected with a sterile blade in wedge shape including base and
margin of ulcer along with wound swab from the same site. Purulent collections are
obtained by aid of needle aspiration. Samples were transported to the laboratory with in an
hour in sterile containers. The materials were subjected to
WET MOUNT

PREPARATION WITH 10% KOH

A small part of the specimen is transferred on to a clean microscopic slide. 2-3

drops of 10% KOH is put on it and the preparation is examined immediately, 10 minutes
and 30 minutes after. On detection of any fungal elements, it was reported.

GRAM STAINED SMEAR EXAMINATION

This was specifically meant for detection of bacterial agents though fungal
elements also can be detected by this method.

CULTURE OF THE MATERIAL

Ulcer materials (pus, tissue material) were streaked daily on to a freshly
prepared and dried SDA and SDA with antibiotic. SDA plates were then incubated for a
period of one week.

 The growth obtained on SDA were studied for

 Rate of growth
 Colony morphology
 Pigmentation of colony and medium
 CHARACTERISATION AND IDENTIFICATION OF DIFFERENT
FUNGAL ISOLATES:

MICROSCOPIC CHARACTERISTICS

LACTOPHENOL COTTON BLUE:

Lacto phenol cotton blue (LPCB) is used to study the morphological features of the
fungal isolates.

Small amount of fungal growth is transferred to a drop of Lacto phenol cotton

blue (LPCB) on a clean glass slide and teased apart with dissecting needles. The Lacto
phenol cotton blue kills, preserves and stains the fungal specimen. A cover slip is applied
and the specimen is examined microscopically under low magnification and then to high
magnification. The main disadvantage of this method is that the characteristic
arrangement of spores is disrupted when pressure is applied to the cover slip.
Under microscope the following features were noted.

 Mycelium –whether true or pseudo mycelium

 Hyphae –whether septate or non septate
 Whether branching or not
 Whether pigmentation present or not
 Spore bearing structures

SLIDE CULTURE

Identification of fungus was also done by the slide culture technique. It was
performed whenever the LPCB mount was found to be insufficient for diagnosis. This
method might appear to be the most suitable for making the microscopic identification of
an organism because it allows one to observe microscopically the fungus growing directly
underneath the cover slip.
  Cut a small block of suitable agar medium in 4x4 mm thickness
 Place the agar block over a sterile glass slide in a Petri dish
 With a right angled wire, inoculate the four quadrants of agar block with organism.
 Apply a sterile cover slip on to the surface of the inoculated agar block.
 Add small amount of sterile distilled water and incubate at room temperature.
 After a suitable incubation period, remove the cover slip and place it on a micro
slide containing a drop of Lacto phenol cotton blue.
 Observe microscopically for the characteristic shape and arrangement of spores.
Fungi were identified according the morphological guidelines of Conat and Smith
1971, Rippon 1988 and Koneman 1997.

The samples which had shown growth of yeast (Candida sp) were first identified by
germ tube test and in parallel they were also streaked on to CHROM agar Candida and
incubated at 370 c without carbon dioxide in the dark for one week. After incubation The
results were read according to colour and morphology of colonies. The species
identification was done as per manufacturer’s instruction and previous reports (Odd’s et
al,1994) ( Beighton et al,1995) .
FUNGAL COLONY MORPHOLOGY SPECIAL TEST
ISOLATES
MACROSCOPY MICROSCOPY

GERM TUBE TEST

Candida sp
The culture suspended
in normal pooled human
CREAM YEAST CELL sera was incubated at
COLORED PSEUDO HYPHAE 370 C for 2-4 hours. A
drop of suspension
SMOOTH AND SEEN
examined for long tube
PASTY like projections extending
from yeast cells called
germ tubes

Aspergillus sp Colonies are Uniseriate/Biseriat

velvetty , yellow e phialides yellow
to green or to green coloured
brown conidia,
Reverse:Golden conidiophores are
to red brown of variable length,
rough pitted and
spiny. The
philalides are
single and double
cover entire vesicle
point out in all
directions
Fusarium sp Greyish white MICROCONIDIA
colonies 1-2 celled,
brownish produced from long
pigment on lateral philiades
reverse arising from
laterally borne
conidiophores
MACROCONIDIA
1-5 septate in
inequilaterally
fusoid with widest
point above the
centre
 COLONY MORPHOLOGY ON CHROM agar Candida sp
Candida
CONVEX CREAMY LIGHT TO MEDIUM GREEN C.albicans
CONVEX CREAMY DARK BLUE TO METALLIC C.tropicalis
BLUE
CONVEX CREAMY YEAST COLONIES IN SHADE C.parapsilosis
OF PINK ,LAVENDER TO IVORY

REAGENTS USED
KOH
 Potassium Hydroxide 10 gm
Glycerol 10 ml
Distilled Water 80 ml

LACTOPHENOL COTTON BLUE STAIN [LPCB]

Melted phenol 20 ml
Lactic acid 20 ml
Cotton blue 0.05 gm
Glycerol 40 ml
Distilled Water 20 ml

MEDIAS USED
SABOURAUD’S DEXTROSE AGAR [SDA]
Peptone 10 gm
Dextrose 40 gm
Agar 20 gm
Distilled water 1000 ml
pH 5.6

SABOURAUD’S DEXTROSE AGAR WITH ANTIBIOTICS

Peptone 10 gm
Dextrose 40 gm
Agar 20 gm
Distilled water 1000 ml
Antibiotic 500mg Actidione in 10ml
acetone
H
p 5.6

CHROM AGAR CANDIDA

CHROMOPEPTONE 10 gm
Glucose 20 gm
Chromogenic mix 2 gm
Chloramphenicol 0.5 gm
Agar 15 gm
Distilled Water 1000 ml
pH 6.1
 OBSERVATION AND RESULTS

Of the 50 diabetic foot ulcer cases studied, 16 cases showed Fungal growth (Table
4) Candida sp was the commonest fungal isolate ( 69%) in the study followed by
Aspergillus sp (19%) and Fusarium sp(12%) (Table; 5; Graph: 5 )

A study of age wise distribution of these 16 patients were done (Table 1) in which
65-70 age group showed highest rate of isolation (Graph1 ).Male patients had a highest
rate of isolation than female (Table 2; Graph 2 ).All patients had show a random glucose
level greater than 200 mg/dl.

The study also highlight the significance of duration of diabetes, in which patients
with duration of more than 10 years showed highest rate of isolation (Table 3;Graph3 ).
 Sterile ulcer cases (2%) other fungal, bacterial agents (66 %) that grew in culture
media were not taken in to consideration in this study (Table 4).

As the number of Candida sp was found high, the study made an attempt to
specieate Candida obtained in 11 positive cases(Table 6)for rapid and appropriate
treatment decision in which Candida albicans (45 %) was the most common Candida sp
isolated followed by Candida parapsilosis (36%) and Candida tropicalis (8%) .

DISCUSSION

The purpose of present study we assess the incidence of fungal etiology of foot
ulcers in diabetic patients. Foot infections are major cause of morbidity in people with
diabetes. Devitalized tissue is the where the micro organisms responsible for non healing
ulcers inflict damage. Numerous investigations have been carried on the bacteriology of
diabetic foot (Bemberger et al, 1985); (Gerding et al, 1995); (Lipsky et al, 1990) ;
( Peterson et al, 1989); (Wheat et al, 1935). There are few reports on the incidence of
fungal pathogen in diabetic foot infection (Bader et al, 2003);( Cooper et al, 1997); (Lai et
al, 1993); (Missoni et al, 2005); (Seema Nair et al, 2006). Most reports have described low
incidence of fungal isolation.
 A total number of 50 diabetic foot ulcer cases presented in the DIABETES
HOSPITAL, CALICUT from July 2010-September 2010 were studied ,among them 16
cases are clinically suspected diabetic foot ulcer cases caused by fungus which were
further analyzed.

The age group wise break up was examined, a marginal increase incidence was
observed in age group between 65-70 years. This may be due to the combined effect of
predisposing factors and diseases that may prevalent in older age group along with the
still maintained outdoor activities that predisposes ulcer development and fungal
infections. More over on average, elderly persons (age 65+) with diabetes have more
physiological impairments to healing so that fungus can easily establish. Same
observation was also made by Ekta Bansal et al in a study conducted on diabetic foot
ulcer in Govt:medical college and hospital, Chandigarh.

The study showed almost equal incidence between both sexes but males were
predominant. Smoking in males is one of the most important features that predispose
infection. It may damage blood vessel in feet and that can disrupt the healing process and
get infected. More over males are more involved in outdoor activities and have high
chance of getting cracks and injury in skin that paves the way to entry of fungus. Similar
observation was made by Missoni et al in University Department of vascular surgery in
Zagreb during his work on incidence of Candida sp in diabetic foot ulcer over a period of
three year, and also by Gopi Chellan et al,2009 in a study conducted on prevalence of
fungal infections in patients with type2 diabetes mellitus in AIMS , Kochi.

The present study also compared the relationship between fungal infections of
diabetic foot ulcer and duration of diabetes, in which a high incidence was shown by in
patients with duration of more than 10 years. Impaired circulation in all organs is the
consequence of long term diabetes with age as an additional contributory factor. In
patients with an history of long term duration the major complications of diabetes i.e.
neuropathy and ischemia can decrease the viability of skin and nails and impair the normal
immune response thus increasing susceptibility to infection .The same observation was
also made by Lavery et al,1998.Loss of protective sensation in long term diabetics render
foot more vulnerable to trauma and coupled with decreased tissue viability increases the
likelihood of a breech in skin integrity and fungal infections (Caroline McIntosh, senior
lecture in podiatry, University of Huddersfield ,Yorkshire).

The study detected fungus as the etiologic agent of diabetic foot ulcer in 16 cases
(32%). A high incidence of mycotic infection in diabetic foot tissue which signified the
need for mycological evaluation of non healing diabetic foot ulcer was shown
earlier(Seema Nair et al,2006). Similar observation was made by Gopi Chellan et al in a
study conducted to detect the prevalence of fungi infecting deep tissues of lower limb
wounds in patients with type 2 diabetes, fungi was found in 27.2% out of which Candida
predominates.

In the present study the most isolated fungus was Candida sp (11 cases;69%). This
high incidence of Candida sp in diabetic foot ulcer was reported earlier by Chinkolikar and
Pal, 2002, and also by McCarty et al, 1994.Various Candida sp in diabetic foot ulcers was
also reported by Mission etal in a study conducted at University hospital Zagreb. The
second most isolated fungus in the present study were Aspergillus sp(19%0) and
Fasarium sp (12%). The same observation was made by [Badee , et al ,2003] and [lai etal
,1993] .Reyes and Rippon have reported cases of simultaneous aspergillosis and
mucurosmycosis complicating diabetic foot ulcer.

Fasarium sp which shared the third position is a mold, has emerged as a pathogen
in immunocompromised patients especially in those under long term steroid therapy.
Fusarium solani, Fusarium moniliformis was observed in non healing diabetic foot ulcer by
various investigators (Ramakrishna Pai et al , 1996. Mustafa Ozurt et al, 2008) isolated
Fusarium sporotrichoides from a diabetic foot ulcer patient with a history of enjoying
walking bare foot.

The study also focused on the incidence of various Candida sp in fungal diabetic
foot ulcer cases in which primary isolation was made on SDA. This showed a high
incidence of C albicans (45%) followed by C parapsilosis(36%)and C tropicalis(18%). The
same incidence of various species was also observed in a study conducted by Seema nair
et al in 74 patients with diabetic foot infection, in podiatry surgery division, AIMS. They
made a differentiation of species according to various biochemical reaction such as sugar
fermentation , sugar assimilation , tetrazolium reduction, urease etc.This study made use
of a selective media, CHROM agar Candida, which is employed for isolation and
presumptive identification of yeast and filamentous fungi and differentiation of C albicans,
C tropicalis and C krusei (beghton et al 1995).it is developed by a Rambach with the
inclusionof the colonies of C albicans C tropicalis and C krusei produce different clours
thus allowing the direct detection of yest species on the isolation plate .Gopi`chellan et al
reported a high incidence of C parapsiloss followed by C tropicalis and C albicans in a
study conducted in AIMS Kochi. M.Huppert and Caziiinjr reported a high incidence of C
albicans in diabetic foot infection following antibiotic therapy in the year 1955.The need for
routine use of CHROM agar Candida medium for presumptive identification of Candida
yeast species was shown by Jean Philippe et al 1996. Candida albicans appeared as
green colored colonies. It has been observed that beta –N acetyl Galactosaminidase
which was produced by C albicans enables to utilize the chromogenic substrate to be
incorporated in to medim and the isolates of these species were seen as green coloured
colonies. This medium allowed the presumptive identification of additional species such as
C.tropicalis (metallic blue colonies),C.parapsilosis(pink coloured colonies producing a
widest range of colours and morphologies).Identification of yeast pathogens by traditionl
methods requires several days and specific mycological medias.

Amphotericin B, Fluconazole and Caspofungin are currently the antifungal agents

most commonly used to treat candidal infection . This practice supported by FDA approval
and the most recent infectious Diseases Society of America guidelines.

Early reporting on the direct microscopy was found to be highly beneficial

regarding the treatment outcome. Wet mount examination of the ulcer material is found to
be very useful in the early diagnosis and it is found to be a rapid, reliable superior
technique of laboratory diagnosis. Moreover patients with a high blood glucose and a high
glycated heamoglobin levels had significantly more fungal infections and also deep
seated fungal infections is high in diabetic patients. In the context of delayed wound
healing and amputation rates, it is important to study the pathogenicity of fungi in diabetic
foot ulcer and their possible contribution to delayed wound healing .The role of antifungal
agents in ulcer management to be evaluated further. This study signifies that routine
bacteriologic diagnosis of diabetic foot ulcer should be supplemented with targeted
mycologic and histopathologic methods to prevent non healing diabetic foot ulcers and a
prudent antifungal treatment based on culture results rather than depending on broad
spectrum antifungal for cure.

CONCLUSION

Fungi seldom behave as a pathogen in normal host but occur in

immunocompromised host with or without underlying pathogenesis. In patients with
diabetes mellitus foot infections is common ranging from chronic bacterial or fungal
infections to serious limb threatening ones. Peripheral vascular disease, neuropathy,
frequent infections direct adverse effect on host defense mechanism make the patient
especially make vulnerable to infection. The antibiotic treatment given without any
mycological diagnosis significantly increases the rate of growth of fungus. Laboratory
diagnosis is essential for the etiological agent of diabetic foot ulcer because fungal and
many bacterial clinical pictures may be have considerable overlapping. The result of the
investigations largely depends on the method of sample collection and procedure. Deep
tissue samples obtained from the base and margin of ulcers is found to be the best
specimen. Different classification and grading systems provide useful information’s
regarding the depth and about various infectious agents in different layers of deep tissues.
Foot ulcers leads to various complications which ultimately lead to amputation and death.
Foot ulcers are caused by both mold and yeast. The prompt detection and presumptive
identification of the isolated yeast may be an aid for rapid appropriate treatment decisions
in light of differences in susceptibility of the yeast sp to antifungal agents. Proper diabetic
foot care prevents many complications. The etiology behind every non healing diabetic
foot ulcer even after antibiotic treatment is can be fungus. Routine bacteriologic diagnosis
of diabetic foot ulcer should be supplemented with targeted mycological diagnosis and a
prudent antifungal treatment is necessary to prevent life and limb threatening infections.

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