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7

2. REVIEW OF LITERATURE

All forms of diabetes, both inherited and acquired, are characterized by

hyperglycemia, a relative or absolute lack of insulin, and the development of diabetes

specific micro-vascular pathology in the retina, renal capillaries and peripheral nerves.

Diabetes is also associated with premature and accelerated atherosclerotic macro-

vascular disease affecting arteries that supply the heart, brain, and lower extremities.

Pathologically, this condition resembles macrovascular disease in non-diabetic patients,

but it is more extensive and progresses more rapidly in diabetic subjects. As a

consequence of the micro-vascular pathology, diabetes mellitus is now the leading cause

of new blindness in people with 20 to 74 years of age and end-stage-renal-disease

(ESRD). People with diabetes mellitus are the fastest growing group of renal dialysis

and transplant recipients. The life expectancy of patients with diabetic ESRD is only 3 to

4 years.

More that 60% of diabetic patients are affected by neuropathy, which include distal

symmetrical polyneuropathy (DSPN), mono-neuropathies, and a variety of autonomic

neuropathies causing erectile dysfunction, urinary incontinence, gastroparesis, and

nocturnal diarrhoea. Accelerated lower extremity arterial disease in conjunction with

neuropathy makes diabetes mellitus accounting for ~50% of all non-traumatic

amputations globally. The risk of cardiovascular complications is increased by two-fold

to six-fold in subjects with diabetes. Overall, the life expectancy is about 7 to 10 years

shorter than for peoples without diabetes mellitus because of increased mortality from

diabetic complications (Skyler, 1996). Large prospective clinical studies show a strong

relationship between glycemia and diabetic microvascular complications in both type 1

diabetes and type 2 diabetes (DCCT1993, UKPDS 1998). There is a continuous, not

linear, relationship between level of glycemia and the risk of development and

progression of these complications.

2.1. Epidemiology of Diabetes Mellitus.

2.1.1. World Scenario.

Diabetes mellitus is one of the most common chronic diseases in nearly all countries,

and its prevalence is on increase as changing lifestyles lead to reduce physical activity,

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8

and increased obesity. It was described as the “global epidemic” of the 21st century; the

increasing incidence of diabetes will place considerable strain on resources and will

bring suffering to many, if the preventive measures are not put into effect [Apelqvist et

al., 2008]. There have been several previous estimates of the prevalence of diabetes

(King and Rewers, 1993; Amos et al, 1997; King et al, 1998; Wild et al., 2004). The World

Health Organization (WHO) estimated that in the year 2000, roughly 3% of the total

world population had diabetes (these results include type 1 and type 2, but not

gestational diabetes) [Armstrong et al., 1998]. WHO also estimated for the years 2000

and 2030, using data from 40 countries but extrapolated to the 191 WHO member

states only (Wild et al., 2004). Other estimate has been produced by the International

Diabetes Federation (IDF) [IDF 2003; IDF 2006]. The latest estimates of the prevalence

of diabetes in the year 2010 by Whiting et al., (2011) were the data from all the 216

countries of the United Nations. They considered only 91 countries, in which prevalence

studies have been undertaken, 5 of the 10 world’s highest national prevalence occurred

in the Middle-East (Table 1), although only Saudi Arabia (18.7%) is among the 80 most

populous country. The Gulf States have prevalence similar to that of Saudi Arabia, and

20 of the 22 Earthquake Model of the Middle East (EMME) regional countries have

prevalence above the world 2010 prevalence of 6.4%.

Table 1: Top 10 countries for diabetes prevalence in 2010 and 2030. (Adopted

from Shah et al., 2010).

2010 2030 Country Prevalence

(%) Country Prevalence (%)

1 2 3 4 5 6 7 8 9 10

Nauru United Arab Emirates Saudi Arabia Mauritius Bahrain Reunion Kuwait Oman Tongo Malaysia

30.9 18.7 16.8 16.2 15.4 15.3 14.6 13.4 13.4 11.6

Nauru United Arab Emirates Saudi Arabia Mauritius Bahrain Reunion Kuwait Oman Tongo Malaysia

33.4 21.4 19.8 18.9 18.1 17.3 16.9 15.7 14.9 13.8

Only includes countries where surveys with blood glucose were undertaken for that country

In developing countries, the majority of people with diabetes are in the 45 to 64 year

age range (King et al., 1998). In contrast, in developed countries, it was >64 years of age.

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9

By 2030, it is estimated that the number of people with diabetes >64 years of age will be

>82 million in developing countries and >48 million in developed countries. In the

current estimates, on the advice of local experts, the prevalence of diabetes in rural

areas is assumed to be one-quarter that of urban areas for Bangladesh, Bhutan, India,

the Maldives, Nepal, and Sri Lanka (Ramachandran et al., 1999).

2.1.2. Indian Scenario.

The first national study on the prevalence of type 2 diabetes in India was done between

1972 and 1975 by the Indian Council Medical Research (ICMR-New Delhi) (Ahuja, 1979,

Ramachandran et. al., 1988). A National Rural Diabetes Survey was done between

1989 and 1991 in different parts of the country’s rural populations which showed

diabetic prevalence as 2.8 per cent (Sridhar et. al., 2002). The prevalence of 6.1 percent

in individuals aged above 40 years was unexpectedly high at that time for rural area

with low socio-economic status and decreased health awareness (Rao et. al., 1989). In

Kashmir vally, a cross-sectional population survey was done in 2000 and the prevalence

of ‘known diabetes’ was 1.9 per cent among adults aged >40 years (Zargar et al., 2000).

In The National Urban Diabetes Survey (NUDS), a population based study was

conducted in six metropolitan (Delhi, Mumbai, Kolkata, Chennai, Hyderabad, Bangalore)

cities across India and recruited 11,216 subjects aged 20 years and above,

representative of all socio-economic strata (Ramachandran et al., 2001). This study

reported the age standardized prevalence of type 2 diabetes as 12.1 per cent. Another

study conducted in western India showed age-standardized prevalence of 8.6 per cent

in urban population (Gupta et al., 2003). The Amrita Diabetes and Endocrine

Population Survey (ADEPS) (Menon et al., 2006), a community based cross-sectional

survey done in urban areas of Ernakulum district in Kerala has revealed a very high

prevalence of 19.5 per cent.

Prevalence of Diabetes in India Study (PODIS). A multicentric cross sectional

population survey was undertaken to determine the prevalence of diabetes mellitus and

impaired glucose tolerance in subjects aged 25 years and above in 77 centres (40 urban

and 37 rural) across India . A total of 18363 (9008 males and 9355 females) subjects

were studied. 10617 (5379 males and 5238 females) were from urban areas and 7746

(3629 males and 4117 females) from rural areas. The prevalence rate for DM in the total

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10

Indian, urban and rural populations was 4.3, 5.9 and 2.7%, respectively. The

corresponding IGT rates in the three populations were 5.2, 6.3 and 3.7%, respectively.

The urban prevalence of DM and IGT was significantly greater in this study than in the

rural population (P < 0.001 in both instances). The prevalence of DM was significantly,

more than that of IGT (P < 0.001) within both the rural and urban populations (Sadikot

et al., 2004).

The latest estimates of the prevalence of diabetes in the year 2010 by Shah et al., (2010)

were accepted by United Nations and published by International Diabetic Federation

(IDF). Estimating more than 50 million people having diabetes in India in 2010, and

figure to increase to more than 87 million with an annual increase of 1813 thousand

people if the preventive measures were not taken to combat this menace (Table 2).

Table 2: Top 10 countries for numbers of people aged 20–79 years with diabetes

in 2010 and 2030. (Adopted from Shah et al., 2010).

2010 2030 Country No. of adults with

Diabetes (millions) Country No. of adults with

Diabetes (millions) 1 2 3 4 5 6 7 8 9 10

India China USA Russian Federation Brazil Germany Pakistan Japan Indonesia Mexico

50.8 43.2 26.8 9.6 7.6 7.5 7.1 7.1 7.0 6.8

India China USA Russian Federation Brazil Germany Pakistan Japan Indonesia Mexico

87.0 62.6 36.0 13.8 12.7 12.0 11.9 10.4 10.3 8.6

2.2. Epidemiology Diabetic Foot Ulcer (DFU) in people with Diabetes.

Foot problems in diabetic patients account for more hospital admissions than any other

long-term complications of diabetes and also result in increasing morbidity and

mortality. The diabetic foot syndrome encompasses a number of pathologies, including

diabetic neuropathy, peripheral vascular disease, Charcot neuroarthropathy, foot

ulceration, osteomyelitis, and the potentially preventable end point amputation.

Patients with the diabetic foot can also have multiple diabetic complications and caring

for such patients may require attention to many different areas.

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The reported rate of foot ulcer prevalence was as high as 11.6 % by Centres for Disease

Control and Prevention (CDCP) (2003) in United States. The 10.6% prevalence rate of

DFU was reported in USA in a population based study, with 2.2% an annual rate on

incidence (Moss et al., 1992). In the same year, 7.4% prevalence rate of DFU was

reported by Walter et al., 1992 in United Kingdom. Other reported rate of incidence was

1.8% in South Asia and 2.7% in African Caribbean and 5.5% in White Europeans (Abott

et al., 2005). The average risk of foot ulcer development in peoples with diabetes is

estimated to be 15% (Palumbo & Melton, 1995). The annual incidence of population

based studies has been summarized in table 3.

Table 3: Summary of selected population based studies estimating incidence and

prevalence of diabetic foot ulcers:

Study (country)

Country Population based

Annual Incidence (%)

Prevalence (%)

Ulcer definition

Method of ulcer asscessment

Abott et al., 2005

United Kingdom

Registered T1 & T2 in 6 UK districts

N=15,692

- 5.5% white european, 1.8% South Asian, 2.7% African Caribbean

Wagner grade ≥1 foot lesion

Clinical examination+chart review

CDCP USA US BRFSS - 11.8% Foot sore that did not heal for >4 weeks

Random digital telephonic

Kumal et al., 1994

UK 3 UK cities

N=811 - 5.3% Wagner

grade ≥1 foot lesion

Direct examination by expert

Moss et al., 1992

USA Population based study

N=1834

2.2% 10.6% NA Medical history questionaire and 4 year later

Muller et al., 2002

Netherland Registered T2DM (1992-1998)

N=3827

2.2% - Full thickness skin loss on the foot

Abstracted medical record

Ramsey et al., 1999

USA Registered T1DM & T2DM (1992-1995)

N=8905

1.9% - Ulcer of lower leg

Medical record

Walter et al., 1992

UK Registered patient from 10 hospitals

N=1077

- 7.4% Wagner grade

Direct examination

Gadepalli et al., 2006

India T1DM & T2DM, Hospital based

N=80

- - Wagner grade

Direct examination by expert

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2.3. Location of foot ulcer in diabetes, its outcome and time to outcome.

The site of ulcer, etiology and treatment methodology varies according to the

population of different region. Dorsal or plantar site are the most common site of ulcer

in diabetic patients followed by plantar metatarsal heads and the heel (Apelqvist J et al.,

1989, Reiber GE et al., 1998). Ulcer severity is more important than site in determining

the final outcome (Apelqvist J et al., 1989). A research conducted by Oyobo et al., (2001),

8% of ulcer was unhealed in their conclusion in a 6-18 months follow-up study. The

details of site of ulcer and its treatment were shown in table 4. Studies conducted by

Margolis DJ., (2000), Margolis DJ., (2002), Pecoraro RE et al., (1991) and McNeely and

Boyko, (2005) reported that several factors were contributing to the outcome of DFU,

which is irrespective of similar type of care treatment. Oyibo et al., (2001) found that

the ulcer surface area were strongly significant in ulcers that healed, did not heal and

proceeded to amputation. The larger size of ulcer will take longer duration of healing

time. After pooling all the five studies, Margolis and colleagues reported that the

neuropathic ulcer took longer duration to heel, 20 weeks for ulcer <2cm2 (Margolis DJ.,

2000). Sex, age and HbA1c level had no impact on the healing of ulcer in a multivariate

analysis model. In medical record of 72,525 diabetic foot wounds from 81,106 patients

from 38 US states, it was conformed that the ulcer/wound that were older, larger in size

and higher in foot grade (≥3) were more likely to have higher duration of healing time

after the age and sex were adjusted (Oyibo et al., 2001). Patients in all these studies

were receiving the similar ulcer care, including off-loading, wound debridement and

healing treatment (Margolis, 2000, Apelvqist et al., 1993, Margolis, 2002, Sheehan et al.,

2003, Pecoraro et al., 1991).

2.4. Risk Factors for Diabetic Foot Ulcer.

The independent risk factors for foot ulcer are demographic variables, risk factors for

foot, health findings and history, health care and education.

2.4.1. Demographic Variables.

The national wide Behavioural Risk Factors Surveillance System (BRFSS), demographic

variables from 2000-2002 for diabetic peoples with or without foot ulcer in a

population based non-institutionalized adults over age of 18 years, self reported foot

ulcer prevalence of 13.7% in a age group 18-44 years, followed by 45-64 years (13.4%),

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13

65-74 years (9.6%) and those with over 75 years (9%). Similar prevalence of foot ulcer

were reported in males and females (11.8% and 11.9% respectively) and this increased

to 9% in patients with duration of diabetes <6years and 19% in those with duration of

diabetes > 21 years (CDCP, 2003). Using the same data, the relative Odds ratio of foot

ulcer was compared with ethnic group, as reference. Compared to Asians, the Relative

Odds ratio were 1.5, with African Americans (OR 2.8), Hispanic (OR 4.2), Native

Americans (OR 7.4) and in Whites (OR 1.8) (McNeely and Boyko, 2005). Abbott and

Colleagues reported the prevalence rates, Europeans (5.5%), South Asians (1.9%), and

African (2.7%). Asian men & women had similarly low foot ulcer rates, few of the

patients were on the insulin; and significantly fewer Asian had ever smoked (Abbott CA.,

2005).

Table 4: Anatomical Locations of Diabetic Foot Ulcer in three prospective studies:

All Ulcersa (N=314)

Most severe Ulcerb (N=302

All Ulcers followed 6-18 monthsc (N=194)

Ulcer Site Toe(dorsal & planter) 51 52 - Planter metatarsals heads, midfoot & heel

28 37 -

Dorsum of foot 17 11 - Multiple ulcers 7 NA - Forefoot - - 78 Mid-foot - - 12 Hind-foot - - 10 Total 100 100 100 Ulcer Outcome Unhealed - - 16 Reepithelialization/primary healing 63 81 65 Amputation at any level 24 14 15 Death 13 5 3.5 Total 100 100 100

a: Apelqvist et al., 1989, b: Reiber et al., 1998, c: Oyibo et al., 1993.

2. 4.2. Foot Risk Factors.

Various risk factors for foot ulcer include peripheral neuropathy, peripheral arterial

disease, and foot deformity. Several quantitative and semi-quantitative methodology

are used for peripheral neuropathy and peripheral arterial studies and these are

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14

summarised in table 05. Both baseline vibration perception threshold (VPT) and

combined score of reflexes and muscle strength are significant predictors of ulcer

(Abbott et al., 2005). Later, a 2.03 relative risk of development of ulceration were found

in patients who were to be detected by 5.07 (10-g) Semmes-Weinstein Monofilament,

which is a semi-quantitative assessment method (Boyko et al1999, Boyko et al., 2006).

Kastenbauer et al., (2001) reported that the elevated levels of VPT greater than 24 volts

as significant risk factors for foot ulcer. While Moss et al., 1992 did not show any

significant association between peripheral neuropathy and foot ulcer. The presence of

two or loss of the four pedal pulses, either with or without the presence of oedema,

indicate the presence of PVD (Abbott CA et al., 2002). A significant correlation between

absence of pedal pulse or history of peripheral arterial revascularization with foot ulcer

have been reported by Kumar et al., (1994), Walter et al., (1992) and Boyko EJ et al.,

(2006). Only a single study reported that foot deformity as a significant risk factor

(Abbott CA et al., 2002) for diabetic foot ulcer.

2. 4.3. Health Findings and History.

Clinical history includes longer duration of diabetes, high HbA1c, prior history of foot

ulcer amputation and smoking habit, a significant relation were reported in between

these and development of ulcer (Kumar et al., 1994, Rith-Najarian et al., 1992, Walter et

al., 1992 and Moss SE et al., 1992). The elevated levels of HbA1c were significantly

associated with development of foot ulcer with an Odds ratio of 1.6 for every 2%

increase in A1c level (Moss et al., 1992) and OR 1.10 for every 1% increase in A1c level

(Boyko et al., 2006). Smoking is one of the significant factor associated with the DFU in a

cohort study (Moss et al., 1992) while it was non-significant as reported by Kastenbauer

et al., (2001), Kumar et al., (1994), Abbott et al., (2002), Boyko et al., (2006) and Moss et

al., (1999). A significant risk of association was also observed with prior history of foot

ulcer by Abbott et al., (2002) with an OR 2.57. Kumar et al., 1994 also showed similar

pattern with an OR 12.7 for subsequent amputations.

2.4.4. Health Care and Education.

Health care and education aere also reported to be an important risk factor for foot

ulcer. Abbott et al., (2002) in a cohort study showed an elevated risk of 2.19 for prior

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15

attendance at podiatric clinic. This variable is most likely an intermediary in pathway of

foot ulcer development.

Table 5: Risk factors for foot ulcers in patients with diabetes mellitus from final

analysis models.

Foot Findings Health and health findings

Author, type of analysis

Study design, type of diabetes

Neuropathy (monofilament, reflex, vibration, or neurological summary) L

ow

AA

I o

r A

bse

nt

Pu

lse

De

form

ity

Lo

ng

D

ura

tio

n

Hig

h H

bA

1c

Sm

ok

ing

Ulc

er

LE

A

Abbott et al., 1998. Cox regression analysis

RCT, patients with VPT ≥25 (US, UK, Canada) T1DM: 255, T2DM: 780

0 Monofilament +VPT +Reflex

Exc

lusi

ve

crit

eria

0

Exc

lusi

on

cr

iter

ia

Exc

lusi

on

cr

iter

ia

Abbott et al., 2002. Cox regression analysis

Cohort, UK registered patients of 6 districts, T1+T2=6613

0VPT +Monofilament +NDS +Reflex

+ + 0 0 +

Boyko et al., 2006. Cox proportion hazards

Cohort, 1285 veterans

+

Dat

a n

ot

incl

ud

ed

0 0 + 0 + +

Carrington et al., 2002. Cox regression analysis

Cohort, single UK clinic, T1DM:83 T2DM:86 No DM:22

+Motor neuropathy 0 VPT 0 Pressure 0 Thermal E

xclu

siv

e cr

iter

ia 0 0 0

Exc

lusi

ve

crit

eria

Kastenbauer et al., 2001.

Cohort, T2DM: 187

0 Monofilament +VPT E

xclu

siv

e cr

iter

ia 0 0 0 0

Exc

lusi

ve

crit

eria

E

xclu

siv

e cr

iter

ia

Kumar et al., 1994. Logistic regression

Cross sectional T2DM: 811 from UK

+ NDS + + 0 0 +

Litzelman et al., 1993. GEE

RCT, T2DM:352

+ Monofilament

0 0 0 +

Exc

lusi

ve

crit

eria

Moss et al., 1992. Logistic regression

Cohort, 2990 DM

+ +

Yo

un

g

Rith-Najrian et al., 1992. Chi sq test

Cohort. T2DM:358 Indians

+Monofilament 0 +

Walters et al., 1992. Logistic regression

Cohort. UK T1+T2:1077

+ Absent light touch. + Impaired pain, perception. 0 VPO

+ A

bse

nt

pu

lse.

0

Do

pp

ler

+ 0

AAI: ankle are index, DM diabetes mellitus, LEA: lower limb amputation, NDS: neuropathic disability

score, RCT: randomozed controlled trials, VPT: vibration perception threshold. Blank cell: not studiesd, +:

statictically significant, 0: non-significant.

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2.5. Epidemiology of Lower Limb Amputation.

2.5.1. Incidence, Prevalence & Amputation.

The amputation rate varies across the geographical locations within the country and or

within the countries. The Global Lower Extremity Amputation Study Group first time

reported the rate of amputation in lower limb in diabetic subjects, pooling a data from

the 10 study centres over a period of 2 years in USA. They reported a lowest rate 2.8 per

100,000 persons per year in Madrid, Spain and the highest in Navojo population with a

rate of amputation as 43.9 per 100,000 per year (Glease, 2000). The annual incidence of

amputation has been shown in table 06. The annual incidences of amputation were

changes from 0.7 per 1000 in East Asia to 31.0 per 1000 in USA (Moss SE et al., 1999,

Humphrey et al., 1966, Humphrey et al., 1994, Lehto et al., 1996, Trautner et al., 1996).

Lower Extremity Amputation (LEA) rates can be 15 to 40 times higher among the

diabetic versus non diabetic populations (ADA 1996; Larvey et. al., 1996; Frykberg,

1999; Resnick et. al., 1999, Viswanathan and Kumpatla, 2011). It was estimated that

every 30 second, amputation was done due to diabetes somewhere in the world

(Viswanathan and Kumpatla, 2011). It was also suggested that, amputation was done

for disease management and remained a global problem for all persons with diabetes

(Jefficoate, 2005). The same risk factors that predispose to ulceration can also generally

be considered contributing causes of amputation, albeit with several modifications.

Another frequently described risk factor for amputation is chronic hyperglycemia.

Results of the Diabetes Control and Complications Trial (DCCT) and the United Kingdom

Prospective Diabetes Study (UKPDS 1998) support the long held theory that chronic

poor control of diabetes is associated with a host of systemic complications (DCCT

1993; UKPDS 1998). The best predictor for amputation is a history of previous

amputation. A past history of a lower extremity ulceration or amputation increases the

risk for further ulceration, infection, and subsequent amputation (Harris, 1998; Larsson

et. al., 1998; Boyko et. al., 1999; Adler et. al., 1999). It may also be inferred that patients

with previous ulceration possess all the risk factors for developing ulceration, having

demonstrated that they already have the component elements in the causal pathway

(Reiber et. al., 1995; Frykberg, 1998; Larvey et. al., 1998). Up to 34% of patients develop

another ulcer within 1 year after healing an index wound, and 5 year rate of developing

a new ulcer is 70% (Apleqvist J et al 1993; Larsson et. al., 1995). Re- amputation can be

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attributed to disease progression, non-healing wounds, and additional risk factors for

limb loss that develop as a result of the first amputation. Tragically, the 5 year survival

rate after a diabetes related LEA has been reported to be as low as 28% to 31% (Ebskov,

1998). One study reported a 5 year mortality rate of 68% after lower limb amputation,

with lower survival rates in those patients with higher levels of amputation. Persons

with renal failure or more proximal levels of amputation have a poor prognosis and

higher mortality rate. Those who undergo a diabetes- related amputation have a 40% to

50% chance of undergoing a contralateral amputation within 2 years (Tentolouris,

2004).

Table 6. Age adjusted population based amputation incidence rates among

patients with diabetes from selected studies.

Author Population studies Annual incidence/1000 Chaturvedi et al., 2001

T1DM: American Indians, Cuban, European, East Asian. T2DM: American Indians, Cuban, European, East Asian.

31.0 8.2 3.5 1.0 9.7 2.0 2.5 0.7

Humphrey et al., 1996 Nauru 7.6 Humphrey et al., 1994 Rochester, USA 3.8 Letho et al., 1996 East & West Finland 8.0 Morris et al., 1998 Tayside Scotland 2.5 Moss et al., 1999 Wisconsin, USA

Young onset diabetic Older onset diabetes

5.1 7.1

Muller et al., 2002 T2DM, primary care, Netherland 6.0 Nelson et al., 1998 Pima Indians, USA 13.7 Siitonen et al., 1993 Incident LEA,

Finland 3.4 Men 2.4 Female

Trautne et al.,1996 Germany 2.1 Van Houtum and Lavery, 1996

California, USA Netherland

4.9 3.6

2.5.2. Risk factors for non-traumatic lower limb amputation in peoples with

diabetes.

Population based study of diabetic individuals with non-traumatic amputations from

the US hospitals were the three main well known geographical risk factors: age, gender

and non-white racial status. In the year 2003, there was an increase in amputation rate

by 2 fold in those who were at the age of ≥75 years (CDCP). The importance of quality of

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care, easily assessable care clinic and socio-economic of patients were also important

significant factors in deciding the rate of amputations (Nielsen et al., 1998, Resnick et

al., 1999, Abbas et al., 2005).

The peripheral neuropathy is also associated with the amputation risk. These includes

insensitivity to 10g monofilament, decrease motor nerve conduction velocity of the

deep peroneal nerve and sensory nerve conduction velocity of the sural nerve, VPT,

absent or diminished bilateral vibration sensation, and absent Achilles tendon and

patellar reflexes. Table 07 shows the studies that reported a statistically significant

association between one or more measures of peripheral neuropathy and amputation

(Lehto et al., 1996,Nelson et al., 1988, Reiber et al., 1992, Selby and Zhang, 1995, Adler

et al., 1999, Hamalainen et al., 1999, Selby et al., 2001, Mayfield et al., 1996, Adler et al.,

1999, Hamalainen et al., 1999, Mayfield et al., 1996, Hennis et al., 2004, Selby et al.,

2001). While Lee et al., (1993), Moss et al., (1999), Resnick et al., (2004) did not show

any association with the amputation and peripheral neuropathy.

The peripheral arterial function, as measured by absent or diminished dorsal pedis and

posterior tibialis pulses as well as median arterial calcification and its relationship to

amputations, is an independent risk factors to predict the amputation in diabetic

patients with foot ulcer (Lehto et al., 1996, Nelson et al., 1988, Reiber et al., 1992, Adler

et al., 1999, Hamalainen et al., 1999, Mayfield et al., 1996, Hennis et al., 2004, Resnick et

al., 2004). High blood pressure is also an independent predictive risk factor for

amputation in various studies (Moss et al., 1999, Selby and Zhang, 1995, Lee et al.,

1993). Six studies have reported no significant role of high blood pressure with

amputation rate in their final outcome (Lehto et al., 1996, Nelson et al., 1988, Reiber et

al., 1992, Hamalainen et al., 1999, Mayfield et al., 1996, Hennis et al., 2004). High BP was

the significant factor in males and elevated diastolic BP in females for amputation (Lee

et al., 1993).

Long duration of diabetes is also significantly associated with amputation (Moss et al.,

1999, Lehto et al., 1996, Nelson et al., 1988, Selby and Zhang, 1995, Adler et al., 1999,

Hamalainen et al., 1999, Selby et al., 2001, Mayfield et al., 1996, Lee et al., 1993, Hennis

et al., 2004, Resnick et al., 2004). Poor glycemic control as measured by elevated HbA1c

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with or without plasma glucose reported to be an important risk factors for increased

risk of amputation in a nine analytical studies represented in table 7. Smoking was an

independent risk factor for increased risk of amputation in diabetic patients (Moss et al.,

1999) and also the patients with history of prior foot ulcer, an independent risk factor

(Moss et al., 1999, Adler et al., 1999, Mayfield et al., 1996). The retinopathy and its

association are also represented in table 7. There is a statistical significant association

between the risk of amputation and presence of retinopathy (Moss et al., 1999, Lehto et

al., 1996, Nelson et al., 1988, Reiber et al., 1992, Selby and Zhang, 1995, Hamalainen et

al., 1999, Mayfield et al., 1996, Lee et al., 1993).

2.6. Neuropathic Problems of the Lower Limb in Diabetic Mellitus.

2.6.1. Definition, Pain Overview & Stages of Diabetic Neuropathy

Definition: The American Diabetes Association has proposed the recommendation on

classification, diagnosis, and treatment guidelines of diabetic neuropathy. They

reiterated the definition as “the presence of symptoms and/or sign of peripheral nerve

dysfunction in people with diabetes after the exclusion of other causes” (Boulton et al.,

1998). Clinical neuropathy is confirmed by the presence of abnormal neurological

examination done by physicians who were skilled in these techniques.

Pain Overview: Mayo C et al., (1931) said that “of all the symptoms for which

physicians are consulted, pain in one form or another, and is the most common and

extremely urgent”. It is very important to understand that diabetic neuropathy can

occur with no pain or with insensate foot or may present with pain in the form of

dysesthesias and paraestheisias. 15% of patients of diabetic patients with neuropathy

have been reported to be suffering from pain (Dyck et al., 1993). Patients may have non-

neuropathic pain or nociceptive pain or both. Nociceptive pain arises from a stimulus

outside the nervous system and is proportionate to receptor stimulation. In acute

nociceptive pain, it serves as protective function. The neuropathic pain arises from

primary lesion or dysfunction in the nervous system and is disproportionate to receptor

stimulation (Serra J, 1999). Neuropathic pain does not require nociceptive stimulation,

and there is often other evidence of nerve damage.

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Table 7: risk factors for Non-traumatic Lower limb amputations in patients with Diabetes Mellitus from Final analysis models

of selected studies.

Author, Analysis type

Stu

dy

de

sig

n

Ty

pe

of

dia

be

tes

Foot findings Health and health histoty

Ne

uro

pa

thy

(m

on

ofi

lam

en

y,

vib

rati

on

, re

fle

x,

NC

V)

PA

D A

AI,

MA

C,

TcP

O2

, Pu

lse

s

HB

P

Du

rati

on

Hig

h H

bA

1c,

FP

G

Sm

ok

ing

Ulc

er

Re

tin

op

ath

y

Adler et al., 83 Multivariate proportional

hazard Cohort 776; T2DM +

+ 0

0

0

+

Hamalainen et al., 84 Logistic regression Nested Case control: 100 +

+

0

+

0

Hennis et al., 88 Logistic regression Case control 309 DM +

+

0

0

+

0

0

Lee et al., 87 Cox regression cohort 875 T2, Oklahama

Indians

+SP

B♂

+

DB

P♀

+

+♂

0

0

+

Lehto et al., 1996 Cox regression Cohort 1044 T2, Finland +

+

0

+

+

0

Mayfield et al., 86 Logistic regression Retrospective case control 246 T2 Pima Indians +

+

0

+

+

0

+

+

Moss et al., 54 Logistic regression Cohort 2990 early & late

onset

+D

BP

+

+

+ Y

ou

nge

r

+

+

Nelson et al., 63 Stratified Cohort 4399 pima Indians +

+

0

+

+

0 +

Reiber et al., 80 Logistic regression Prospective case control 316 T1DM +

+

0

Co

ntr

ol

var

iab

le

+

0 +

Resnick et al., 131 Logistic regression Cohort

+A

BI

>4

.1

OK

=+

P

ima=

0

+

+

0

Selby and Zhang 81 Logistic regression Nested retrospective case

control 428 T1+T2 +

+SP

B

+

+

0 +

AAI:ankle arm index, DBP:diastolic blood pressure, FPG: fasting plasma glucose, HbA1c: haemoglobin A1c, HBP: high blood pressure, MAC: medial arterial

calcification, NCV; nerve conduction velocity, PtEd:patient outpatient education, PVD; peripheral vascular disease, SBP: systolic blood pressure, TcPo:

transcutaneous oxygen tension, Blank cell:not studies, +: significant finding, 0: non-significant findings.

Review of Literature

21

Stages: The Mayo clinic staging methods is useful one to describe the different stages of

diabetic neuropathy (Dyck PJ, 1988). The different stages are shown in table 8.

Table 8. Different stages of Diabetic Neuropathy.

Stage Description Sign/ Symptoms

Abnormal quantitative test

0 No Neuropathy No No

1 Subclinical Neuropathy No Yes

2 Clinical evident Neuropathy Yes Yes

3 Debilitating Neuropathy Yes Yes

2.6.2. Classification of Diabetic Neuropathy.

Diabetic neuropathy can be classified in several ways as: clinical presentation

(symmetrical, focal, or multifocal or painful, paralytic, and ataxic), predominant type of

fibers affected (motor, sensory, autonomic), or painful or nonpainful. Dyck et al., (1987)

gave the classification of diabetic neuropathy, presented in table 09.

Table 9: Classification of diabetic neuropathy.

Symmetrical distal neuropathy

Symmetrical Proximal neuropathy

Asymmetrical proximal neuropathy

Cranial

Trunk radiculopathy or mononeuropathy

Limb plexus or mononeuropathy

Multiple mononeuropathy

Entrapment mononeuropathy

Ischemic nerve injury from acute arterial occlusion

Asymmetrical neuropathy and symmetrical distal neuropathy

Various types of neuropathy have been described in terms of their onset, symmetry, and

clinical course (Table 10). A symmetrical distal neuropathy is the most common type

with symptoms of numbness, tingling, and burning in the lower feet and lower shins or

it can be asymptomatic detected by neurological examination or electrophysiological

testing.

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22

Table 10: Types of Diabetic Neuropathy.

Focal neuropathy Ischemic neuropathy

o Sudden onset

o Asymmetrical

o Ischemic etiology

o Self limited

o Examples

Mononeuropathy

Femoral neuropathy

Radiculopathy

Plexopathy

Cranial neuropathy

Entrapment neuropathy

o Gradual onset

o Usually asymmetrical but can bi bilateral

o Compressing etiology

o Waxing and waning progression without spontaneous recovery

o Examples

Carpal tunnel syndrome

Ulnar entrapment (tennis elbow)

Lateral cutaneous femoral nerve entrapment

Tarsal tunnel syndrome

Diffuse neuropathy

o Insidious onset

o Symmetrical

o Abnormal secondary to vascular metabolic syndrome, structural and autoimmune aberration.

o Progression without spontaneous recovery

o Example.

Distal symmetrical poly-neuropathy

Autonomic neuropathy

2.6.3. Risk Factors for Diabetic Neuropathy.

There are two man risk factors for the diabetic neuropathy, non-modefiable and

modifiable. The detail overviews are presented in table 11. In a modifiable factor, only

the hyperglycemia has been proven to be a significant risk factor via prospective,

randomized, multicentric, parallel design clinical study (ADA-1995). Whereas other

factors listed in the table are significant by retrospective or cross sectional data study

only. The non-modifiable factors include older age, longer duration of diabetes, HLA

DR3/4 genotype and patients height. Many studies confirm that, male sex are at greater

risk but on reanalysing the data, it shows that, it’s because of the greater height of male

Review of Literature

23

than female and hypothesized that longer nerve are more prone to nerve damage

(Robinson et al., 1992, Sosenko et al., 1986).

Table 11. Risk Factors for Diabetic Neuropathy.

Non-modifiable risk factor o Old age o Longer duration of diabetes o HLA DR3/4 genotype

o Greater Height

Modifiable risk factor

o Hyperglycemia o Hypertension o Elevated Cholesterol Level o Smoking o Heavy alcohol use

2.6.4. Pathogenesis of Diabetic Neuropathy.

In the most diabetic complaints, insulin deficiency, and hyperglycemia are the major

factors involved in both type of diabetes (type 1 and type 2). In this regard, a multiple

retrospective study which support this hypothesis was DCCT trial (DCCT-1993) and

UKPDS (Thomas, 1973, Dyck, 1987) that are presented in table 12., which is the

strongest evidence supporting this mechanism in both type of diabetes.

Table 12: DCCT & UKPDS Landmark Clinical trials of Glucose control.

Variables DCCT UKPDS

Type of diabetes patients Type 1 Type 2

Number of patients 1441 4209

Study length (yrs) 10 20

Length of patient followup (yrs) 5 10

Average HbA1c (%)

Standard therapy 8.9 7.9

Intensive therapy 7.1 7.0

Average glucose level (mg/dl)

Standard therapy 231 117

Intensive therapy 155 147

Reduction in retinopathy (%) 76 21

Reduction in Nephropathy (%) 56 34

Reduction in Neuropathy (%) 60 25

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24

Both the trials were the randomized prospective trials comparing groups of intensive

treatment with conventional therapy to achieve the tight glucose control. In both these

trials, the group of patients who were on intensive treatment have better nerve

conduction velocity and furthermore, it also confirm that, patients with tight glucose

control had less autonomic neuropathy.

The pathogenesis of diabetic neuropathy includes various factors, like hyperglycemia

(DCCT-1993; Thomas, 1973; Dyck, 1987), pre-diabetes neuropathy (Genuth et al.,

2003,Borch-Johnson 2001, Zhang et al., 2003, Polydefkis et al., 2003, Alexendrea et al.,

2003, Haslbeck et al., 2005), Vaso Nervorum (Beggs et al., 1992, Cameron and

Cotter,1997, Johnson and Beggs, 1993), Protein Kinase C pathway activation (Xia et al.,

1994, Greene and Lattimer, 1985, Cole et al., 1995, Danis et al., 1998, Hata et al., 1999),

abnormal fatty acid metabolism (Cameron & Cotter, 1996, Cameron et al., 1996), Polyol

pathway (Cameron et al., 1997, Goldfarb et al., 1991, Obrosova et al., 1997), Myo-

inositol (Yorek et al., 1993), advanced glycated end product (Friedman, 1999, Honing et

al., 1998), antibody to neural tissue (Canal & Nemni, 1997, Jaeger et al., 1997, Zanone et

al., 1998), nerve growth factor (Vinik et al., 2005, Pitterger & Vinik, 2003). In the year

2004, four major pathogenic pathways mechanism explaining the hyperglycaemic nerve

damage (Brownlee, 2005). These four major mechanisms are increased intracellular

formation of AGEs, increased polyol pathway, activation of protein kinase C and

increased hemosamine pathway flux.

One theory is that due to chronic hyperglycemia in diabetes there is accelerated non-

enzymatic glycation of intracellular proteins resulting in the formation of Advanced

Glycation End (AGE) products by the so called Maillard reaction (Maillard, 1912). In

normal individuals, glucose reacts with amino groups to form some amount of Early

Glycosylation products (stable Amadori products) through a nonenzymatic and

irreversible process. It has also arisen from intracellular auto-oxidation of glucose to

glyoxal, decomposition of the Amadori product to 3-deoxygluconase, and fragmentation

of glyceraldehyde-3-phosphate to methylglyoxal. These reactive intracellular

dicarbonyls react with amino groups of intracellular and extracellular proteins to form

AGEs. Different mechanisms have been explained by which AGE precursors have

deleterious effects on target tissues. AGE act as recognition signals for uptake and

Review of Literature

25

degradation of proteins by macrophages resulting in the production of cytokines, and

growth factors like TNF-a, IL-1, IGF-1, GM-CSF, G-CSF, and PDGF and ultimately

oxidative stress. Modification of important matrix proteins like type 1 collagen, type 4

collagen, and laminin also occurs, resulting in change in structure and function of

extracellular matrix most importantly reduction in endothelial cell adhesion. AGE

modified proteins can also alter cellular function through binding to specific AGE

receptors (RAGE) on endothelial cells. RAGE belongs to a class of immunoglobulin super

family is expressed on the surface of a variety of cell types, including endothelial cells,

smooth muscle cells, lymphocytes, monocytes, and neurons. Activation of RAGE triggers

the generation of reactive oxygen species and further activation of signaling molecules

such as nuclear factor kB , p21, and PKC (Figure 1).

Figure 1: Increased production of AGE precursors and its pathologic consequences. From Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820, 2001.

Normally intracellular glucose is predominantly phosphorylated to Glucose 6 phosphate

by the enzyme Hexokinase to be metabolized later on by glycolysis and HMP shunt and

only some amount is converted to sorbitol by the Polyol pathway. Enzyme Aldose

reductase is the rate limiting step in this alternate pathway and it has a low affinity for

glucose. Under conditions of hyperglycemia there is increased flux of glucose through

this pathway that is about 30% and is metabolized in this way. In a hyperglycemic

environment, however, increased intracellular glucose results in increased enzymatic

conversion to the polyalcohol sorbitol, with concomitant decreases in NADPH. The

Review of Literature

26

mechanism by which glucose flux through the polyol pathway is detrimental and not

clearly defined. It has been proposed that oxidation of sorbitol by NAD+ increases the

cytosolic ratio of NADH/NAD+, there by inhibiting activity of enzyme glyceraldehydes-

3-phosphate dehydrogenase and increasing concentratons of triose phosphate. Elevated

triosephosphate concentratons could increase formation of both methylglyoxal, a

precursor of AGE and diacylglycerol , thus activating PKC (Brownlee M 2001). It has

also been found that reduction of glucose to sorbitol by NADPH consumes the cofactor

NADPH which is required for regenerating reduced glutathione (GSH). This can lead to

oxidative stress as glutathione is an important cellular antioxidant. Some other

proposed mechanisms are sorbitol induced osmotic stress and decreased Na+,K+-

ATPase activity. But the sorbitol concentration in diabetic vessels and nerves are too

low to be considered significant (Brownlee M et al 2003) (Figure 2).

Figure 2: Hyperglycemia increases flux through the polyol pathway. From Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820, 2001.

The third important mechanism is the activation of family of Protein Kinase C

enzymes which are cell signaling enzymes. These enzymes are involved in diverse

cellular functions ranging from cell growth and differentiation, apoptosis, protein

trafficking, cytoskeletal rearrangement, and cell polarity. PKC isofoms are activated by

second messenger Diacyl glycerol. In states of hyperglycemia, there is increased

concentration of DAG as a result of its denovo synthesis from glyceraldehyde-3-

phosphate. Activation of PKC has been associated with suppression of nitric oxide (NO)

Review of Literature

27

production via inhibition of insulin stimulated expression of endothelial nitric oxide

synthase (eNOS) and stimulation of endothelin 1(ET-10) vasoconstrictor activity. It also

leads to increased microvascular matrix protein accumulation by inducing the

expression of TGF-β1, fibronectin, and type IV collagen. It has also been implicated in

the over expression of the fibrinolytic inhibitor PAI-1 and in the activation of pleiotropic

transcription factor NF-kB. It also induces expression of the permeability-enhancing

factor VEGF (Figure 3).

Figure 3: Consequences of hyperglycemia-induced activation of PKC.

Figure 4: Hyperglycemia increases flux through the hexosamine pathway. From Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820, 2001.

A fourth hypothesis is that, in states of hyperglycemia there is increase flux of glucose

through Hexosamine pathway. Fructose-6-phosphate which is derived from glucose is

ultimately converted to UDP-N-acetylglucosamine (UDP-GlcNAc). This product

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28

glycosylates the threonine and serine residues of target proteins by the enzyme UDP-

GlcNAc, which can alter the functional properties of many transcriptional regulatory

factors (Figure 4).

All these were seemed to reflect single hyperglycaemic induced process of

overproduction of superoxide by michondrial electron transport chain (Canal, 1997).

These associations might help in to explain the detail mechanism involved in the

metabolic syndrome to develope diabetic neuropathy before they are diagnosed as

overt type 2 diabetes (Figure 5).

Figure 5: Mitochondrial overproduction of superoxide activates four major pathways of hyperglycemic damage by inhibiting GAPDH. From Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820, 2001.

2.6.5. Documentation of Neuropathy.

The documentation of neuropathy involves the following three major steps

2.6.5.1. Clinical presentation

2.6.5.2. Neurological examination

2.6.5.3. Electrophysiological testing.

2.6.5.1. (i). In the clinical presentation, the major documentation includes type of

pain, motor sign & symptoms, and autonomic neuropathy symptoms.

Review of Literature

29

Types of pain: there are three type of pain as described by David Ross (Pfeifer et al.,

1993). (a) Dysesthesis has been attributed to cutaneous and subcutaneous and it may

be attributed to increased firings of damage or abnormal nociceptive fibers. (b)

Paraesthesis pain occurs from several factors like spontaneous activity near the cell

body of damage afferent axone in dorsal root ganglion, loss of segment myelinated

fibers, ectopic impulse generation from demyelinated patches of myelinated axon and

last by increased firings. (c) Muscular pain is believed to be secondary caused by injury

to motor neuron (e.g. demyelinated patch). The details of type of pain are shown in

table 13. (ii). Motor sign and symptoms: This symptom relates with the loss of pro-

prioception in toes and ankle, a sign that is commonly used in testing of joint position

senses in feet. Leg weakness is typically a late feature of diabetic neuropathy where as

ankle weakness is typically the first motor symptoms of diabetic neuropathy. (iii) In

Autonomic Neuropathy, the patients usually complain dry and cracked skin, orthostatic

dizziness & urinary retention, etc.

Table 13. Descriptors of Type of Pain in Neuropathy.

Dysestheis Burning sensation, sunburn type, skin tingles, painful sensation when something touches.

Paraesthesis Pins and needles like, electric shock like, Numb but achy, feel like ice water shock, shooting pain, lancinating pain

Muscular Pain Dull ache, night cramp, band like sensation, drawing sensation, deep ache, spasms.

2.6.5.2. In the neurological Examination, following are the testing for diabetic

neuropathy. Monofilament Testing: on the plantar surface of the great toe and the pulp

of the index figure bilaterally to assess the sense of touch in booth feet. The currently

available instrument is Semmes Weinstein Monofilament usually made from fine nylon

fibre and is designed to give an appropriate pressure to the site. Each monofilament is

calibrated to deliver different bowing force which is represented in the form of number

that represents the log decimal 10 times the force in milligram ranging from

1.65(0.45gm) to 6.65(447gm) of linear force. The patients inability to sense the force of

monofilament 5.07 (10 gm) was considered as neuropathic positive. The second

documentation is Deep Tenson Reflex which is an essential part of examination

Review of Literature

30

because the deep tendon reflex are reduced or absent in length dependent pattern such

as ankle lost first than knee (Sharma et al., 2002). The third documentation is motor

function, which is most common as many patients will have no demonstrable weakness

in feet. Once the neuropathy advances to knee, patients begin to complaints about the

hand also.

2.6.5.3. Electrophysiological Testing. It plays an important role in evaluating both

types of neuropathies in patients (suspected and well documented). It defines that

which fibre is affected (motor, sensory and autonomic), renders a gross estimate of the

duration of neuropathy and even gives insight of the progression also. The

electromyography can be used to examine the de-nervation and to decipher chronicity,

and later by analysing the morphology of motor nerve.

2.7. Peripheral Vascular Disease in Diabetes.

The studies of various risk factors which provide the insight into the pathogenesis of

peripheral vascular atherosclerosis in diabetes have been already discussed in PAD.

Factors like hypertension and cigarette smoking are also a predictive significant risk

factor for PVD (Colwell, 1986). A strong correlation among them in relation to PVD has

been well established in various geographical locations Rochester, Minnesota (Palumbo

et al., 1991), Seattle, Washington (Beach et al., 1979), Munich, Germany (Janka et al.,

1980), Kuopio, Finland (Uusitupa et al., 1990) and Framingham, Massachusetts (Garcia

et al., 1974). Hyperglycemia was not an independent risk factor for PVD in a study on

populations of Japan (Colwell, 1986). While, in USA and Germany, fasting glucose and

HbA1c values are the good predictors of PVD. In a large scale studies, certain lipid-

lipoproteins changes have been reported to be a risk factor for PVD in diabetes. The

decrease level of HDL-C and elevated serum triglycerides were reported to be the

possible risk factor for PVD in cross-sectional study on 252 diabetic subjects (Beach et

al., 1979). In Finland, increased plasma cholesterol, VLDL cholesterol, HDL-C

(Biesbroeck et al., 1983), increased VLDL, LDL-C levels (Uusitupa et al., 1990).

Hyperinsulinemia emerged as independent risk factor in many epidemiological studies

(Fontbonne et al., 1991). The epidemiological studies suggest that, the lipids,

lipoproteins may, in particular, contribute to PVD as well as hypertension, smoking, and

hyperglycemia. Data from the Indian subcontinent regarding PVD, among patients are

Review of Literature

31

very scarce. The prevalence of neuropathy was 75% in a study conducted by Gadepalli

et al., (2006) in North India. In The Chennai Urban Population Study (CUPS)

(Premalatha et al., 2000), the overall prevalence rate of PVD in enrolled subjects was

3.2%, 2.7% in patients with normal glucose tolerance group, and in impaired glucose

tolerance group it was 2.9%. In the type 2 diabetic patients, the prevalence of PVD in

newly diagnosed diabetic was 3.5% and 7.8% in known diabetic subjects. Whereas, the

overall PVD prevalence was 6.3% which was higher from overall subjects enrolled in

CUPS study. The prevalence of PVD in India is comparable to that report from Sri Lanka

(Weerasuriya et al., 1998) but is considerably lower than that reported from Rochester

study (Melton et al., 1980) and the Hoorn Study (Beks et al., 1995). This suggests that

different susceptibility factors may operate in different populations. Alternatively, it

could also be because the prevalence of certain well-known risk factors, e.g., smoking,

could be less common in certain populations. Finally, it could simply be a reflection of

the younger age structure of the population, as shown by a steep increase in prevalence

rates of PVD in those patients >70 years of age. The differences between the prevalence

rates of CAD and PVD in CUPS study are quite striking. Thus, whereas CAD occurs with

increased prevalence and at a younger age (premature CAD), PVD appears to show the

opposite trend, i.e., lower prevalence and occurrence in older age-groups. This finding

suggests that the pathogenic mechanisms for CAD and PVD could be different. In

addition, CUPS results suggest that screening for CAD using the ABI (Federman et al.,

1998, Kornitzer et al., 1995) is unlikely to be useful in a South Asian population. Indeed,

the risk factors for PVD itself appear to differ in different populations. A study from

China (Cheng et al., 1998) reported that hypertension, diabetes, elevated serum

cholesterol, LDL cholesterol, triglycerides, fibrinogen, and hyperglycemia are associated

with PVD. A U.S. study showed diabetes to be the major risk factor for PVD

(Papademetriou et al., 1998). In Greece, serum triglycerides alone were found to be

associated with PVD in diabetic subjects (Katsilambros et al., 1996). A prospective study

on type 2 diabetes showed triglycerides, HDL cholesterol, hypertension, and smoking as

risk factors for PVD (Beach et al., 1998). Other reports showed microalbuminuria (Jager

et al., 1999), homocysteine (Taylor et al., 1999), and lipoprotein(α) (Wollesen et al.,

1999) to be associated with PVD. However, the commonly known risk factors do not

explain the high prevalence of PVD seen in some ethnic groups (Kannel et al., 1990,

Uusitupa et al., 1990). Although smoking provided a 2.7 times higher risk for PVD, this

Review of Literature

32

did not reach statistical significance. The absence of association with smoking can be

attributed to a small sample size or to the underreporting of smoking because of

cultural and other barriers. Unfortunately, we could not perform serum nicotine

estimation to quantify smoking in this study. The weak association with serum lipids

may also be because of the small sample size.

2.7.1. Pathophysiology of PVD.

Atherosclerosis (also known as arteriosclerotic vascular disease or ASVD) is a condition

in which an artery wall thickens as a result of the accumulation of fatty materials such

as cholesterol. It is a syndrome affecting arterial blood vessels, a chronic inflammatory

response in the walls of arteries, caused largely by the accumulation of macrophage

white blood cells promoted by low-density lipoproteins (plasma proteins that carry

cholesterol and triglycerides) without adequate removal of fats and cholesterol from the

macrophages by functional high density lipoproteins (HDL), (apoA-1 Milano). It is

commonly referred to as a hardening or furring of the arteries. It is caused by the

formation of multiple plaques within the arteries (Maton et al., 1993). Atherosclerotic

lesions or atherosclerotic plaques are separated into two broad categories: Stable and

unstable (also called vulnerable) (Ross & Russel, 1999).

The main cause of atherosclerosis is yet unknown, but is hypothesized to fundamentally

be initiated by inflammatory processes in the vessel wall in response to retained low-

density lipoprotein (LDL) molecules (Williams & Tabas, 1995). Once inside the vessel

wall, LDL molecules become susceptible to oxidation by free radicals (Sparrow &

Olszewski, 1993), and toxic to the cells. The damage caused by the oxidized LDL

molecules triggers a cascade of immune responses which over time can produce an

atheroma. The LDL molecule is globular shaped with a hollow core to carry cholesterol

throughout the body. The body's immune system responds to the damage to the artery

wall caused by oxidized LDL by sending specialized white blood cells (macrophages and

T-lymphocytes) to absorb the oxidized-LDL forming specialized foam cells. These white

blood cells are not able to process the oxidized-LDL, and ultimately grow, then rupture,

depositing a greater amount of oxidized cholesterol into the arterial wall. This triggers

more white blood cells, continuing the cycle. Eventually, the artery becomes inflamed.

The cholesterol plaque causes the muscle cells to enlarge and form a hard cover over

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33

the affected area. This hard cover is what causes a narrowing of the artery, reduces the

blood flow and increases blood pressure.

Some researchers believe that atherosclerosis may be caused by an infection of the

vascular smooth muscle cells; chickens, for example, develop atherosclerosis when

infected with the Marek's disease herpesvirus (Fabricant & Fabricant, 1999).

Herpesvirus infection of arterial smooth muscle cells has been shown to cause

cholesteryl ester (CE) accumulation (Hsu et al., 1995). Cholesteryl ester accumulation is

associated with atherosclerosis.

Atherogenesis is the developmental process of atheromatous plaques. It is

characterized by a remodeling of arteries leading to subendothelial accumulation of

fatty substances called plaques. The build up of an atheromatous plaque is a slow

process, develope over a period of several years through a complex series of cellular

events occurring within the arterial wall, and in response to a variety of local vascular

circulating factors. One recent theory suggests that, for unknown reasons, leukocytes,

such as monocytes or basophils, begin to attack the endothelium of the artery lumen in

cardiac muscle (Figure 06). The ensuing inflammation leads to formation of

atheromatous plaques in the arterial tunica intima, a region of the vessel wall located

between the endothelium and the tunica media. The bulk of these lesions is made of

excess fat, collagen, and elastin. At first, as the plaques grow, only wall thickening occurs

without any narrowing. Stenosis is a late event, which may never occur and is often the

result of repeated plaque rupture and healing responses, not just the atherosclerotic

process by itself.

Early atherogenesis is characterized by the adherence of blood circulating monocytes to

the vascular bed lining, the endothelium, followed by their migration to the sub-

endothelial space, and further activation into monocyte-derived macrophages

(Schwartz et al., 1992). The primary documented driver of this process is oxidized

Lipoprotein particles within the wall, beneath the endothelial cells, though upper

normal or elevated concentrations of blood glucose. Fatty streaks may appear and

disappear. Low Density Lipoprotein particles in blood plasma, when invade the

endothelium and become oxidized create a risk for cardiovascular disease. A complex

set of biochemical reactions regulates the oxidation of LDL, chiefly stimulated by

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34

presence of enzymes, e.g. Lp-LpA2 and free radicals in the endothelium or blood vessel

lining.

Figure 6: New concept about the pathogenesis of atheroscelerosis, with emphasis

on the roles of monocyte derived macrophages, cytokines, immune

complexes, glycation and oxidation of LDL, and endothelial adhesion

proteins.

The initial damage to the blood vessel wall results in an inflammatory response.

Monocytes (a type of white blood cell) enter the artery wall from the bloodstream, with

platelets adhering to the area of insult. This may be promoted by redox signaling

induction of factors such as VCAM-1, which recruit circulating monocytes. The

monocytes differentiate into macrophages, which ingest oxidized LDL, slowly turning

into large "foam cells" – so-described because of their changed appearance resulting

from the numerous internal cytoplasmic vesicles and resulting high lipid content. Under

the microscope, the lesion now appears as a fatty streak. Foam cells eventually die, and

further propagate the inflammatory process. There is also smooth muscle proliferation

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35

and migration from tunica media to intima responding to cytokines secreted by

damaged endothelial cells. This would cause the formation of a fibrous capsule covering

the fatty streak (Figure 7).

Figure 7: A postulate scheme for the mechanism of atherosclerosis in diabetes,

emphasizing the role of glucose in the process.

2.7.2. Risk Factors.

Various anatomic, physiological and behavioral risk factors for atherosclerosis are

known (Blankenhorn & Hodis, 1993). Risks multiply, with two factors increasing the

risk of atherosclerosis fourfold (Mitchell et al., 2007). Hyperlipidemia, hypertension and

cigarette smoking together increases the risk seven times (Mitchell et al., 2007).

(a). Modifiable

Diabetes or Impaired glucose tolerance (IGT) (Mitchell et al., 2007)

Dyslipoproteinemia (unhealthy patterns of serum proteins carrying fats &

cholesterol): (Mitchell et al., 2007)

High serum concentration of low-density lipoprotein (LDL, "bad if elevated

concentrations and small"), and / or very low density lipoprotein (VLDL) particles,

i.e., "lipoprotein subclass analysis"

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36

Low serum concentration of functioning high density lipoprotein (HDL "protective if

large and high enough" particles), i.e., "lipoprotein subclass analysis"

An LDL:HDL ratio greater than 3:1

Tobacco smoking, increases risk by 200% after several pack years (Mitchell et al.,

2007).

Having hypertension +, on its own increasing risk by 60% (Mitchell et al., 2007).

Elevated serum C-reactive protein concentrations (Mitchell et al., 2007, Narain et

al., 2008).

Vitamin B6 deficiency (Rinehart & Greenberg , 1949, Rinehart & Greenberg , 1956,

Gruberg & Raymond , 1981).

(b). Nonmodifiable.

Advanced age, male sex, having close relatives who have had some complication of

atherosclerosis (e.g. coronary heart disease or stroke), Genetic abnormalities,[15] e.g.

familial hypercholesterolemia (Mitchell et al., 2007).

2.8. Biomechanics of the Foot in Diabetes.

Most of the skin injuries on the feet of diabetic patients with neuropathy mainly occur in

forefoot, with equal distribution on plantar and dorsal surface (Edmond et al., 1986),

and those on plantar are frequently occur at site of higher pressure areas (Veves et al.,

1991, Stess et al., 1997). Now various tools are available to measure the pressure areas

under bare foot walking and also inside the shoes. The overall component of brief

examination of foot in two minutes was shown in table 14.

Table 14: A “Two minute foot examination” to check for biomechanical and other risk factors once a patient has been at risk of neuropathy or vascular disease.

Evaluation Component

Look for

1. Surface Exam Ulcer, callus, hemorrhage in callus, blister, maceration between toe, mbreaks in skin, skin infection, edema, erythema, temperature.

2. Nail Exam Fungal infection, ingrown toenail, injury. 3. Foot deformity Prominent metatarsal head, clawed toes or hammertoes, rocker

bottom foot deformity, prior amputations.

4. Examination of shoes

Drainage into socks, flatted insoles, leaning one side, poorly fitted,

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37

One of the important aspects of biomechanics study is the planter pressure

examination. Defined by Armstrong et al., (Armstrong et al., 1998), a pressure of 750kPa

will provide a cut-off value to differentiate between the low risk and high risk patients.

The second most important aspect is foot deformity and the various risk factors for

foot deformity are depicted in table 14, which is the key factor for injury in the foot. The

high foot pressure on dorsal and plantar surface will cause most skin injury. This may

lead to vulnerable areas on the foot predisposing to ulceration. Motor neuropathy, with

imbalance of the flexor and extensor muscles in the foot, frequently results in foot

deformity, with prominent metatarsal heads and clawing of the toes, In turn, the

combination of proprioceptive loss due to neuropathy and the prominence of

metatarsal heads lead to increases in the pressures and load under diabetic foot

(Brownlee et. al., 2003). In the charcot process, the foot is also erythematous, hot, and

swollen, but in the healed stage, these findings are absent. The third aspect is the callus

in the foot. The etiology of callus is still unknown. In a study by Murray et al., (1996), the

callus was present with the loss of sensation in foot, that have high risk of developing an

ulcer. It is therefore, strongly recommended to remove the callus from the foot using

appropriate measures. The third important aspect is the limited joint mobility.

The relationship of joint limitation and plantar ulceration was established in a study by

Delbridge et. al., (1985). There was a significant correlation between joint mobility at

the subtalar joint and mobility at the first metatarsophalangeal joint. Additionally,

Mueller et. al., (2001) found significant decrease in sensation, ankle dorsiflexion and

subtalar joint motion in diabetic patients with ulceration compared to normal controls

They demonstrated the linkage of neuropathy and joint limitation with plantar

ulceration in patients with diabetes. Birke et al., (1988) demonstrated the relationship

of hallux limitus with great toe ulceration. They found significantly decreased great toe

extension using a torque range-of-motion system in diabetic patients with a history of

great toe ulcers compared to diabetic patients with a history of ulcers at other sites and

normal controls. Fernando et al., (1991) found significantly increased foot pressures

using pedobarography in diabetic patients with limited subtalar and

metatarsophalangeal joints compared to diabetic patients and controls without limited

mobility. As shown by these studies, sensory loss and joint hypomobility may result in

increased pressure and plantar ulceration. Orthoses and footwear, designed to spread

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the stresses over time or reduce the function motion requirements (e.g., rocker sole)

during walking time, are needed to compensate for hypomobility in the feet of patients

with hypomobility (Hampton et. al., 1990; Coleman, 1990; Thompson, 1988).

2.9. Classification of Foot Lesions in Diabetes Mellitus.

For evaluation and determination of the severity of diabetic foot, various classification

systems are in use now, that attempt to encompass different characteristics of an ulcer

(namely site, depth, the presence of neuropathy, infection, and ischemia, etc). (Oakley et.

al., 1956; Shea, 1975; Wagner, 1981; Knighton et. al., 1986; Kaufman et. al., 1987; Jones

et. al., 1987; Lavery et. al., 1996) including Wagner System, University of Texas System

and a hybrid System, Depth Ischemic classification , the PEDIS System. It seems that

poor clinical outcomes are generally associated with infection, peripheral vascular

disease, and increasing wound depth; it also appears that the progressive cumulative

effect of these comorbidities contribute to a greater likelihood of a diabetic foot ulcer

leading to a lower-limb amputation.

The three main diabetic foot classification system are discussed that are commonly used

in clinical diagnosis of diabetic foot.

These were:

2.9.1. Wagner-Meggitt Classification

2.9.2. Depth-Ischemic classification

2.9.3. University of Texas classification

2.9.1. Meggit-Wagner Classification.

Most common and widely used Classification system is the Wagner Diabetic Foot

classification System (Table 15). This system is basically anatomical with gradations of

superficial ulcer, deep ulcer, abscess osteitis, gangrene of the fore foot, and gangrene of

the entire foot. Only grade 3 addresses the problem of infection. In this system foot

lesions are divided into different grades starting from grade 0 to grade 5. Grade 0

includes high risk foot but no active lesion and grade 5 includes gangrene of entire foot.

But this system does not mention about ischemia or neuropathy and that is the

drawback of this system.

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Table 15- Wagner-Meggitt Classification System [Wagner FW, 1981].

Grade Lesion 0 No open Lesion 1 Superficial ulcer 2 Deep ulcer to tendon or joint capsule 3 Deep ulcer with abscess, osteomyelitis, or joint sepsis 4 Local gangrene- fore foot or heel

5 Gangrene of entire foot

2.9.2. Depth-Ischemic classification.

This classification is a modification of Wagner-Meggit system. The purpose of this

classification system is to make the classification more accurate, rational, easier to

distinguish between wound and vascularity of foot, to elucidate the difference among

the grades 2 and 3, and to improve the correlation of treatment to the grade. Details of

depth Ischemic classification are presented in table 16.

Table 16- depth Ischemic classification System

Grade Lesion 0 No open lesions: may have deformity or cellulitis A Ischemic B Infected 1 Superficial ulcer A Ischemic B Infected 2 Deep ulcers to tendon, or joint capsule A Ischemic B Infected 3 Deep ulcers with abscess, osteomyelitis, or joint sepsis A Ischemic B Infected

4 Localized gangrene — forefoot or heel A Ischemic B Infected 5 Gangrene of entire foot A Ischemic B Infected

2.9.3. University of Texas Classification system .

Another popular system is the University of Texas San Antonio System which

incorporates lesion depth and ischemia (Table 17). It is actually a modification of

Wagner System .In this system each grade of Wagner System is further divided into

stages according to the presence of infection or ischemia or combination of both. This

system is somewhat superior in predicting the outcome in comparison to the Wagner

System.

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Table 17: University of Texas Classification System [Armstromg DG et al., 1998].

Stages

Grades

0 I II III

A Pre-or post ulcerative lesions Completely epithelialized

Superficial wound not involving tendon capsule or bone

Wound penetrating to tendon or capsule

Wound penetrating to bone or joint

B With infection With infection With infection With infection

C With ischemia With ischemia With ischemia With ischemia

D With infection and ischemia

With infection and ischemia

With infection and ischemia

With infection and ischemia

2.10. Infection Problems of the Foot in Diabetic Patients.

The infection in the foot of diabetic patient is a major medical, social and economic

problem and the leading cause of hospitalization for patients with DFU. The trio of

problem leading to diabetic foot is neuropathy, vascular changes and infection.

Infections of various types may be more common and are more often severe in patients

with diabetes mellitus (Boyko & Lipsky, 1995, Gleckman & Al-Wawi, 1999, Jackson,

2005, Muller et al., 2005). On the basis of result of large retrospective cohort study,

nearly half of all the diabetic patients have atleast one hospitalization in their lifespan

for treatment of infection in Canada (Shah & hux, 2003). The risk was 1.21 times in

diabetic subjects when compared with control group. Foot infections are the most

common in diabetic patients range from relatively mild to limb threatening (or life

threatening). Almost all the infections begin as a minor problem may progress to

involve deep tissue, joints, or bony especially if it was not managed. The infection

complicates the pathological picture of diabetic foot (Apelqvist et al., 2000; Anandi et al.,

2004). The worst and the most feared outcome of infection in DFU is lower limb

amputation (Reiber et al., 1995). Diabetes continues to be the leading cause of lower

limb amputations worldwide. The WHO has estimated that there are approximately

250,000 lower limb amputations per year in diabetic patients in Europe alone (Margolis

et al., 2005).

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2.10.1. Epidemiology of Infection in Diabetic Foot Patients.

Soft tissue and foot infections are significantly associated with diabetes (Boyko &

Lipsky, 1995). The relative frequency of cellulites is 9 times more in diabetic patients

than in nondiabetic subjects (Boyko & Lipsky, 1995). Similarly, patients with

osteomyelitis were more who had infections in foot in their report and the relative

proportions of hospitalization of patients with osteomyelitis were 12 times more than

in non-diabetic subjects (Boyko & Lipsky, 1995). The risk of hospitalization was more

than non diabetic subjects. Significant risk factors that were independent risk factors in

a multivariate analysis were infections to bone and duration of ulcer >30 days and

peripheral vascular disease (Lavery et al., 2006). The variable in 112 diabetic foot ulcer

were history of previous amputation, peripheral vascular disease and neuropathy but

not socioeconomic status as the significant risk factors (Peter et al., 2005). Fortunately,

upto 50% diabetic individual who had one foot infection episode will have another

episode within few years. Thus infection is often the proximate cause leading to

amputation in their outcome (Pecoraro et al., 1991, Reiber et al., 1992). India, with a

population of more than 1.1 billion, has the dubious distinction of having a larger

number of people with diabetes and there were no major difference in the risk factors

when compared with developed countries, while the clinical features may vary in

developing countries because of the regional factors (Pendsey, 1994). Role of Pathogens

in diabetic foot infection in India. In a study, the prevalence of pathogens in diabetic foot

infections in relation to parameters such as Wagner’s grading, duration of diabetes, and

healing times was studied (Viswanathan et al., 2002). It was found that in 654 diabetic

patients, 728 pathogens were isolated. Aerobic pathogens were isolated in 66.8%

patients and anaerobic pathogens were isolated in 33.2%. As Wagner’s grading

increased, the prevalence of anaerobic pathogens also increased.

Neuropathy was once again found to be a common factor in diabetic patients infected

with both aerobic and anaerobic pathogens. Ulcers infected with anaerobic pathogens

showed a longer healing time than ulcers infected with aerobic pathogens (P < 0.001).

Among the frequently discovered aerobic pathogens, the Enterobacteriaceae family was

the most prominent at 48%, the Staphylococcus species was quite prominent at 18.2%,

Streptococcus stands at 16.8% and Pseudomonas at 17%. Among anaerobes

Peptostreptococcus and Clostridium formed 69.4%, Gram-negative anaerobes such as

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Bactericides and Fusobacterium were present in 30.6%. Healing times were longer when

strict aerobic pathogen Pseudomonas and strict anaerobic pathogens were present

(136.1 ± 28.6 and 136.4 ± 34.7 days, respectively). Another study conducted by

Gadepalli et al., (2006), 80 diabetic foot ulcer patients with clinical sigh and symptoms

of infection were studied. Males were predominant (85%), all patients were from

Wagner grade 3-5, majority of the subjects were from type 2 diabetes with mean age

was 53.9 years. The mean duration of diabetes was 11.8 years. The neuropathy was

present in 86.2%, PVD 85%, retinopathy and hypertension in 72%. Among 80 patients,

58 had MDRO infections, 38 has ESBL infection and 6 as MRSA and 8 patients as both

MRSA and ESBL. In their study, PVD, neuropathy, ulcer size (>4cm2), poor glycemic

control, higher grade of ulcer were associated with MDRO infection. The

epidemiological data on diabetic foot worldwide were presented in table 18.

Table 18. Epidemiological data on diabetic foot ulcer worldwide

Country Study n Prevalence Incidence

ulcer amputation ulcer amputation Uk Abbott et al., 2002 9710 1.7 1.3 2.2 - Nauru Humphrey et al., 1996 1564 - - - 0.76 Uk Kumar et al., 1994 811 1.4 - - - Greece Manes et al., 2002 821 4.8 - - - Netherland Muller et al., 2002 665 - - 2.1 0.6 Algeria Balhadj, 1998 865 11.9 6.7 - - India Pendsey, 1994 11300 3.6 - - - Slovenia Urbancic-Rovan & Slak,

1998 701 7.1 - - -

N: total diabetic population.

2.10.2. Pathophysiology.

A variety of physiology and metabolic distributions conspire to place diabetes patients

at high risk for foot wounds. Microbial colonization of wound is inevitable, usually with

endogenous bacteria, but these are potentially pathogenic in wound (Bowler, 2002).

Immunological disturbance are also an important predisposing factors of

pathophysiology of foot ulcer; these includes abnormalities of migration, phagocytosis,

intracellular killing, and chemotaxis. The cellular immune response and monocyte

function are also reduced in diabetes. Poor granulation formation, prolonged abscess

presence and impaired wound healing are further complicating the diabetic foot ulcer.

Once the skin has been breached, continued mobilization on a broken area impairs the

healing process. Inevitably, direct contiguous spread of microbes on the skin follows on,

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with colonization and infection of superficial and then deeper tissues is likely if the

process is allowed to proceed unchecked. Both the healing process and the response to

infection are further compromised by vascular insufficiency, which is commonly

present in patients burdened with complications of diabetes (Andrew et al., 2010). The

infection in diabetic foot is mainly by aerobic bacteria (Anandi et al., 2004; Gadepalli et

al., 2006; Citron et al., 2007; Ramakant et al., 2011). Anaerobic bacterial infection also

plays a significant role in the infection of DFU but this has not been studied since the

strict anaerobic culture techniques are not available at all the clinical laboratories. The

impact of anaerobes was reported first by Louie et al., (1976) and subsequently by

Fieres et al.,(1979); Sapico et al. (1984); Amalia et al., (2002); Gadepalli et al., (2006);

Citron et al., (2007); Lily et al., (2008). There are only few reports available on the

incidence of fungal pathogens in diabetic foot infections [Sapico et al 1980; Cooper and

McGinnis, 1997; Jenkis et al., 2001; Bader et al., 2003; Abdulrazak et al., 2005; Raja et al.,

2007; Bansal et al., 2008]. DFU infection is usually polymicrobial in nature and this was

first reported by Louie et al., (1976), and subsequently by Fieres et al., (1979), Anandi et

al., 2004; Gadepalli et al., 2006; Ramakant et al., (2011) . The unique anatomy of the foot

is the main reason that infection is potentially serious in this location (Rauwerda, 2000,

Armstrong & Lipsky, 2004). The structure compartment, tendons, sheaths, and

neurovascular bundles tend to favour the proximal spread of infection. The deep planter

spaces were divided into medial, central, and lateral compartments. The infections may

spread from one compartment to another at their calcaneal or by direct performation of

septae, but lateral or dorsal spread is a late sign on infection (Bridges & Deitch, 1994).

2.10.3. Microbial Consideration.

2.10.3.1. Definition.

The skin of person is coated with bacteria that are harmless and many of these

organisms are present permanently, but some are transient bacteria that are more often

isolated from skin and soft tissue infection, such as S aureus and beta haemolytic

streptococci which are rarely present on skin. It may be present transiently on the skin

and wounds. When the bacteria multiply in the tissue of diabetic patients, it releases

toxins inciting a host response; the wound is defined as infected. Infection involves the

invasion of host tissue inflammatory response (erythema, indurations, pain or

tenderness, warmth, loss of function, purulent discharge)the number and type of

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organisms, their interaction within each other and with the wound, vascular status , and

host resistance will collectively influence whether or not the wound will heal or become

more infected (Bowler, 2003). This may be due to single organism’s infection called

monomicrobial and more than one organism’s infection called polymicrobial infection.

2.10.3.2. Wound Culture.

Culturing a clinically uninfected wound is unnecessary unless if it is required for the

epidemiological purpose. Defining the microbiological causes in infected foot will

usually assist in subsequent management of infection. A culture of samples will identify

the microbes present in infection, but only if the specimen(s) are collected properly and

processed according to the recommended guidelines. Usually the patients with severe,

long standing diabetes, complicated foot and previous antibiotic use will have

polymicrobial infection in their wound. Culture specimens are sometimes defined by

how results are reliable (Wheat wt al., 1986). A curettage or tissue scraping from the

base of ulcer provides more accurate result (Lipsky et al., 1990). Specimen should be

send to microbiology laboratory for aerobic and anaerobic culture and sensitivity

report. Mild infections in patients who not receive antibiotic therapy may have 1 or 2

bacterial infection in their foot ulcer, whereas serious infection usually caused by

polymicrobial infection (aerobic alone or with anaerobic bacteria) (Sapico et al., 1984).

Anaerobes are rarely the sole pathogen but most often participate in mixed infection

(Dang et al., 2003, Eady & Cove, 2003).

2.10.4. Diagnosis and Assessing Severity.

2.10.4.1. Diagnosing Infection.

Infection should be accessed first by appearance of a local foot problem (e.g. pain,

swelling, ulceration, sinus tract, or crepitation) a systemic infection (e.g. rigor, vomiting,

tachycardia, confusion, malaise, and fever) or a metabolic disorder (severe

hyperglycemia, ketosis, azotemia). It should be considered when the local signs are less

severe than expected (Williams et al., 2004). Proper evaluation of diabetic foot infection

requires critical and methodological approaches which are listed in table 19.

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Table 19: Recommended evaluation of diabetic patients with foot ulcer.

Describe the lesion (cellulites, ulcer, etc) and any drainage (serous, purulent).

Enumerate the presence or absence and degree of various signs of

inflammation.

Define the status of infection and determine the probable cause.

Examine the soft tissue for evidence of crepitus, abcesses, sinus tract, foreign

particle.

Probe any skin break with sterile probe to see whether bone is exposed or not

Measure the size of wound, take photograph

Palpate and record pedel pulse, use Doppler instrument if necessary

Evaluate neurological status

Cleaned and debride the wound

Culture the cleaned wound

Order the plain radiograph

2.10.4.2. Severity of Infection.

Various classification systems were already proposed for diabetic foot infection but

none were universally accepted. Assessing the severity is essential in selecting the

appropriate antibiotics. This will influence the route of drug administration and need

for hospitalization. It will also help in assessing the potential necessary and timing of

surgery. A simple clinical classification of infection has been presented in table 20.

Table 20. Simple Clinical classification of severity of DFU.

Superficial ulcer or cellulitis present

Deep tissue or bone involve

Tissue necrosis or gangrene present

Systemic toxicity or metabolic instability

present

Mild √ - ± - Moderate √ ± (no gas or

fasciitis) ± (minimal) -

Severe √ ± ± √

√=present; ± = may or may not; - = not present

The clinical features that help to define the severity of an infection were represented in

table 21.

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Table 21: Clinical characteristics that help to define the severity of an infection.

Features Mild Infection Serious infection Presentation Slowely progressive Acute or rapidly progressive Ulceration Involves skin only Penetration to subcutaneous tissue Tissue involved Epidermal and dermal Fascia, muscle, tendon, joint, bone.

Cellulitis Minimal (<2cm ring) Extensive, or distant from ulceration Local signs Slight inflammation Severe inflammation, crepitus, bullae.

Systemic signs None or minimal Fever, chills, hypotension, confusion, volume depletion, leukocytosis

Metabolic control Mildly abnormal Severe hyperglycemia, acidosis, azotemia

Foot vasculature Minimal impaired (reduce pulse)

Absent pulse, reduced ankle or toe blood pressure

Complicating features

None or minimal (callus, ulcer)

Gangrene, Escher, foreign body, abscess, marked edema, osteomyelitis.

2.11. Criteria of Infection.

A critical bacterial load, synergic relationship between bacterial species and the

presence of specific pathogens have been proposed as predictors of infection. However,

a critical microbial load might directly affect the healing of both acute and chronic

wounds as first reported by Bendy et al, (1964). Guidelines of The British Association of

Dermatologists and the Royal College of Physicians recommend that infection should be

considered if one of the following is present along with the wound: increased pain,

increasing erythema of surrounding skin, lymphangitis or rapid increase in ulcer size

and pyrexia (Douglas and Simpson, 1995). The Consensus Development Conference on

Diabetic Foot Wound Care (ADA 1999), agreed that a DFU should be considered infected

when there are purulent secretions or there is presence of two or more signs of

inflammation (erythema, warmth, tenderness, heat, induration). Chronic wounds by

their very nature may not always display the classic symptoms of infection (pain,

erythema, oedema, heat and purulence) and it has been suggested that an expanded list,

including signs specific to secondary wounds (such as serous exudate plus concurrent

inflammation, delayed healing, discolouration of granulation tissue, friable granulation

tissue, foul odour and wound breakdown) be employed to identify infection (Gardner et

al., 2001).

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2.12. Antibiotic Therapy.

2.12.1. Route of Therapy.

The antibiotic therapy usually be given intravenously for systemic ill patients, with

severe infection and those who are unable to tolerate oral agents. After a patient

significantly responds to the antibiotic treatment in 3-5 days, most of the patients are

shifted to oral antibiotics (Tice et al., 2003). Oral antibiotic therapy is less expensive and

more convenient. Several newly licensed drugs called fluoroquinolones (Greenberg et

al., 2000, Edmiston et al., 2004) and linezolid (Lipsky et al., 2004) expand the spectrum

of microorganisms that can be treated. Diabetic patients may absorb oral antibiotics

poorly whereas these are well absorbed on oral dosing in nondiabetic patients. For

mildly infected patients, tropical therapy is the better option of treatment. This

treatment has several advantages, including high local drug levels, avoidance of

systemin adverse effect (Lipsky et al., 1997). The patients with PVD, therapautic

antibiotic concentrations with many agents are often not achieved in tissue even while

the serum concentrations are adequate (Raymaker et al., 2001, Legat et al., 2003,

Oberdorfer et al., 2004). In one procedure, called retrograde venous perfusion,

antibiotic solution are injected under pressure into a foot vein while

sphygmomanometer is inflated on the thigh. Recently calcium sulphate beads were used

in the surgical sites and open wound (Armstrong et al., 2003).

2.12.2. Choice of Antibiotic Therapy and Duration.

Many patients will begin therapy, with pending the results of would culture. The narrow

spectrum antibiotics may be used in mild infected ulcers until the report of culture &

sensitivity are received to modify the treatment accordingly, selecting antibiotic agents

that empirically active against Staphylococci and other Streptococci also. The wounds

with foul smell and necrotic and gangrenous usually be treated with anti-anaerobic

antibiotics and later on the treatment will be modified according to the reports. On the

other hand if the infection is not significantly responding to treatment, the treatment

should be changed to cover all the isolated organisms. The antimicrobial spectrum, are

shown in table 22 (a, b, c, d). The necessary duration of antibiotics therapy has not been

well studied. For mild to moderate infections, 1-2 week course are found to be effective

(Lipsky et al., 1990), and for severe, it was 2-4 weeks time. The antibiotic treatment

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should be discontinued when the clinical sign and symptoms of infection have resolved,

even the wound has not completely healed.

Table 22: Selected antibiotics that may be used for diabetic foot infections

(Adopted from Clinical Practice Guideline-2007. Médecine et maladies infectieuses 37

(2007) 14–25).

Table 22 (a) for Enterobacteriaceae

Enterobacteriaceae Molecule Dosage/ 24h Route of

administration Dose interval Comment

Cefotaxime ofloxacin or ciprofloxacin

200 mg/ kg per day 600 mg per day 800–1200 mg per day or 1000–1500 mg per day

IV IV/ Oral IV or Oral

4–6 h 8 h 8 h or 12 h

Oral route as soon as possible

OR ofloxacin or ciprofloxacin

600 mg per day 800–1200 mg per day or 1000–1500 mg per day

IV/ Oral IV or Oral

8 h 8 h or 12 h

Oral route as soon as possible

Table 22 (b) for Streptococcus infection

Streptococcus spp Molecule Dosage/24h Route of

administration Dose interval

Comment

Amoxicilin + rifampicin

150–200 mg/kg per day 20–30 mg/kg per day

IV IV/ Oral

4–6 h 8 or 12 h

Change to oral route as soon as possible IV for first 24–48 hours, then oral route at the physician’s discretion

OR Clindamycinb + rifampicin

1800 mg per day 20–30 mg/kg per day

IV/ Oral IV/ Oral

4–6 h 8–12 h

oral route as soon as possible

OR Vancomycin + rifampicin

1 g (loading dose) then 30 mg/kg 20-30 mg/kg per day

IV IV infusion IV/ Oral

Loading dose (1h) IV infusion or/ 12h 8–12h

Adjust to serum assaysa

OR Teicoplanin + rifampicin

24 mg/ kg per day then 12 mg/ kg per day 20–30 mg/kg per day

IV/ subcutaneous Subcutaneous IV/ Oral

12 h loading dose 24 h 8 or 12 h

For 48 h, then Every 24 ha

a Adjust the dosages to obtain tough concentrations (discontinuous IV) or plateau concentrations (continuous IV) of 30 mg/l for vancomycin, or a tough concentration of 30-40 mg/l by HPLC for teicoplanin b Only if susceptible to erythromycin

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Table 22 (c) for MRSA & MSSA

Methicilin – resistant S. Aureus Molecule Dosage/24h Route of

administration Dose interval Comment

Vancomycin ± gentamicin OR + rifampicin OR + fosfomycin

1 g (loading dose) then 30 mg/ kg 4 mg/ kg per day 20–30 mg/ kg per day 200 mg/kg per day

IV IV infusion IV IV/ Oral IV

Loading dose (1h) IV infusion or/ 12h 24 h 8 or 12 h 8 h

Adjust according to serum assaysa

For 48 h IV for first 24–48 hours, then oral route as soon as possible Infusion over 1–2 h

OR Rifampicin + fusidic acid

20 – 30 mg/ kg per day 1500 mg per day

IV/ Oral IV/ Oral

8 or 12 h 8 h

IV for first 24–48 hours, then oral route as soon as possible

OR [Trimethoprim + Sulfamethoxazole] + rifampicin

640/3200 mg 20–30 mg/ kg per day

IV/ Oral IV/ Oral

(equivalent to 2 tab/12h of [Trimethoprim + Sulfamethoxazole] 8 or 12 h

IV for first 24–48 hours, then oral route as soon as possible

OR Teicoplanin + rifampicin

24 mg/ kg per day 12 mg/kg per day 20–30 mg/ kg per day

IV/ Subcutaneous Subcutaneous IV/ Oral

12 h loading dose 24 h 8 or 12 h

for 48 h, then every 24 ha

a Adjust the dosage to obtain trough concentrations (discontinuous IV) or plateau concentrations (continuous IV) of 30 mg/l for vancomycin, or a trough concentration of 30–40 mg/l by HPLC for teicoplanin

Methicillin – susceptible S. Aureus Molecule Dosage/24h Route of

administration

Dose interval Comment

Oxacillin or cloxacillin = gentamicin

100-15 mg/ kg per day 4 mg/ kg per day

IV IV

4 or 6 h 24 h

Until reception of specimens 4 mg/ kg per day

OR Ofloxacin or perfloxacinb + rifampicin

600 mg per day 800 mg per day 20–30 mg per day

IV/ Oral IV/ Oral IV/ Oral

8 h 12 h 8 or 12 h

Oral route as soon as possible

OR Ofloxacin or perfloxacinb + fusidic acid

600 mg per day 800 mg per day 1500 mg per day

IV/ Oral IV/ Oral IV/ Oral

8 h 12 h 8 h

Oral route as soon as possible

OR Rifampicin + fusidic acid

20–30 mg per day 1500 mg per day

IV/ Oral IV/ Oral

8 or 12 h 8 h

Oral route as soon as possible

OR Clindamycina

+ rifampicin 1800 mg per day 20–30 mg per day

IV/ Oral IV/ Oral

8 h 8 or 12 h

Oral route as soon as possible

OR [Trimethoprim + sulfamethoxazole] + rifampicin

640/ 3200 mg 20–30 mg per day

IV/ Oral IV/ Oral

12 h (equivalent to 2 tab/ 12 h of [Trimethoprim + sulfamethoxazole]) 8–12 h

Oral route as soon as possible

a Only if susceptible to erythromycin b Caution in subjects > 60 years (1/2 dose)

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Table 22 (D) first line antibiotics for Diabetic foot ulcer (excluding osteomyelitis).

First–line antibiotics in diabetes foot infections (excluding osteomyelitis) Type of infection Suspected pathogens Antibiotic therapy Recent infection of a superficial wound (< 1 month)

MSSAb S.pyogenes MRSAc

Cloxacillin or cephalexin or [amoxicillin + clavulanate] or clindamycin Pristinamycin or linezolide or vancomycin or teicoplanin

Extensive cellulitis Deep and/ or chronic lesion with or without sepsis

MSSAb S.pyogenes MRSAc MSSAb

S. pyogenes, GNBd, anaerobes MRSAc MSSAb

S. pyogenes, GNBd, anaerobes MRSAc, GNBd, anaerobes

Oxacillin AGa Vancomycin or teicoplanin or linezolide [Amoxicillin + clavulanate] AGa + vancomycin or teicoplanin or linezolide [Piperacillin + tazobactam] or [ticarcillin + clavulanate] + AGa

Imipenem or ertapenem + [vancomycin or teicoplanin or linezolide] + AGa

Shaded zone: oral outpatient treatment; for the other cases, treatment is initially parenteral, followed by oral therapy when possible, depending on the course and susceptibility profile of the bacteria isolated. a AG: aminoglycosides (gentamicin or netilmicin) b MSSA: methicillin- susceptible Staphylococcus aureus c MRSA: methicillin- resistant Staphylococcus aureus d GNB: Gram-negative bacilli

2.12.3. Outcome of Antibiotic Therapy.

The clinical response to mild and moderate infection can be expected to be 80-90 % and

this rate of treatment output are further reported to decrease to 50-60%, the infections

are of deep or more extensive type, patients usually require surgical intervention in the

form of minor or major. Approximately 2/3 of these patients require amputations in

their feet or one or more bone resection (Van Damme et al., 2001) and long term

outcome are reported to be achieved in 80 % of patients (Armstront & Lipsky, 2004,

Rauwerda, 2004). In many patients, above ankle amputations are avoided and the uses

of aggressive antibiotic therapy with minor and major surgical intervention are

required (Tan et al., 1996). The factors which can predict the ulcer healing includes (i)

absence of exposed bone, (ii) palpable popliteal pulse, (iii) toe pressure of >45 mmHg

and for ankle >80mmHg, and (iv) WBC count <12000 m3 (Eneroth et al., 1997).

2.13. Wound Healing in Diabetic Foot Ulcer.

The healing of diabetic wound is complex mechanism which involves an intricately

regulated sequence of celluler and biochemical events orchestrated to restore tissue

integrity after injury (Figure 08).

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The wound healing process involves the three distinct but overlapping phases (Figure

09):

(i). Hemostasis and inflammation,

(ii). Proliferation &

(iii). Maturation and Remodelling.

Each of these phases is controlled by biologically active agents called growth factors

(Steed, 1997). Growth factors are hormone like polypeptides, present in very small

amounts in the body that control the growth, differentiation and metabolism of cells

(Hunt & Van, 1989).

Figure 8: Normal wound healing is an intricately regulated sequence of cellular and biochemical events orchestrated to restore tissue integrity after injury.

Figure 9: Time sequence of normal wound healing

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2.13.1. Hemostasis and Inflammation.

Hemostasis follows the traumatic rupture of blood vessels exposed to sub-endothelial

collagen and platelets resulting the activation of intrinsic part of coagulation cascade

(Witti & Barbul, 1997). The inflammation is characterized by increased in vascular

permeability, poly-morphonuclear cells (chemotaxis) from circulation into wound and it

activates the migrating cells. The neutrofills are the first to arrive at site of wound and

its role are primary phagocytic and wound debridement and it is a source of

proinflammatory cytokines which serve to activation of the local fibroblast and

keratinocytes (Park & Barbul, 2004, Hubner et al., 1996). It decreases the infection in

wound (Simpson & Ross, 1972), as they are not essential because their role in

phagocytosis and antimicrobial defence are taken up by macrophages. The macrophage

will migrate to into wound 48-98 hrs after injury and participate in inflammatory

process. Their antimicrobial functions will include phagocytosis and generation of

reactive free radicals, such as nitric oxide, oxygen, and peroxide. It develops functional

complement receptors and undertake similar operations to the neurophils (Cherry et

al., 2000). The wound healing is severely impaired if macrophage infiltration of the

wound is prevented.

2.13.2. Proliferation.

The new tissue matrix are essential for physical support for new blood vessel, arising

from the intact vasculature in the surrounding dermis, and angiogenesis is essential to

provide the basic nutrient and oxygen which help in synthesis of collagen, hence

collagen and capillary proliferation occurs in co-dependent manner. During this phase,

fibroblast and endothelial cells are primary cells that multiply in this process. Fibroblast

responsible for replacing the fibrin matrix called clot and also produce and release

proteoglycans and glycosaminoglycans, an extra-component for tissue granulation. The

fibroblast production will stop when sufficient collagen were produced. During healing

and repair, angiogenesis a fundamental biological mechanism is activated (Carmeliet,

2005), which is characterized by invasion, migration and proliferation of endothelial

cells and those which are close to side walls of vessel start migration because of

angiogenesis. The cytoplasmic pseudopods starts sprouting in wound and forms

perivascular space and the endothelial cells also proliferate to cell migration and

produce degradation enzyme (Folkman & Shing, 1992, Clark, 1996). Several biological

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factors like, platelets, macrophages, lymphocyte, keratinocyte production (fibroblast

growth factor α & β, transforming growth factor α & β, epidermal growth factor,

hepatocyte growth factor and IL-1) are reported to be potent stimuli for new vessel

formation (Lynch et al., 1989, Grant et al., 1993). Hypoxia (create oxygen granulation),

lactate and extracellular matrix also mediate cell growth (Knighton & Fiegel, 1989).

Several adhesive protein like Willebrand factor, fibrinogen, fibronectin up regulate the α

& β integrin adhesive receptors. These integrin factors initiate the calcium dependent

signalling pathways leading to cell migration. Once the wound is filled with new

granulation tissue, angiogenesis stops and many vessels disintegrate as a result of

apoptosis.

2.13.3. Maturation and Wound Healing.

The main feature in the maturation phase is collagen deposition in wound space. The

rate of maturation, quality and total amount of matrix deposition determine the

strength of scar (Witte & Barbul, 1997). To support the future matrix deposition and

remodelling, glycosaminoglycans and proteoglycans are synthesized. The collegen is a

predominant scar protein and extracellular matrix components are remodelled by

matrix metaloproteinases. The source of collagenases in the wound is the inflammatory

cells and endothelial cells as well as fibroblastand keratinocyte. These are able to

degenerate and the activity of collagen was tightly controlled by cytokines.

2.14. Pathogenesis and Impact of Microbes on DFU.

The development of a foot ulcer involves the convergence of several pathological

mechanisms (Edmonds, 1986). Diabetic peripheral sensorimotor neuropathy is the key

factor in the majority of cases. As a result of damage to sensory nerves, minor trauma

can go unnoticed. Neuropathy can also deform the architecture of the foot to such an

extent that joints and digits are placed in mechanically unfavourable positions, making

them highly vulnerable to injury. Once the skin is breached, continued mobilization on a

broken area impairs the healing process. Inevitably, direct contiguous spread of

microbes on the skin follows on, with colonization and infection of superficial and then

deeper tissues is, likely if the process is allowed to proceed unchecked. Both the healing

process and the response to infection are further compromised by vascular

insufficiency, which is commonly present in patients burdened with complications of

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diabetes (Andrew et al., 2010). The impact of microbes on wounds has been extensively

studied and reviewed using different approaches to elicit their possible role in healing

process of diabetic foot ulcers. The infection in diabetic foot is mainly by aerobic

bacteria (Anandi et al., 2004; Gadepalli et al., 2006; Citron et al., 2007; Ramakant et al.,

2011). Anaerobic bacterial infection also plays a significant role in the infection of DFU

but this has not been studied since the strict anaerobic culture techniques are not

available at all the clinical laboratories. The impact of anaerobes has been reported first

by Louie et al., (1976) and subsequently studies by Fieres et al.,(1979); Sapico et al.

(1984); Amalia et al., (2002); Gadepalli et al., (2006); Citron et al., (2007); Lily et al.,

(2008). There are only few reports available on the incidence of fungal pathogens in

diabetic foot infections [Sapico et al 1980; Cooper and McGinnis, 1997; Jenkis et al.,

2001; Bader et al., 2003; Abdulrazak et al., 2005; Raja et al., 2007; Bansal et al., 2008].

DFU infection is usually polymicrobial in nature and this was first reported by Louie et

al., (1976), and subsequently by Fieres et al., (1979), Anandi et al., 2004; Gadepalli et al.,

2006; Ramakant et al., 2011.

2.15. Etiology and Prevalence of Infection In DFU.

2.15.1. Global Scenario.

The first report of polymicrobial etiology of DFU infection was from Boston, USA in

1976. On an average, 5.2 organisms infection per patient were reported; which included

3.2 for aerobic and 2.6 for anaerobic organisms respectively. Majority of isolates were

Bacteroides sp (14.6%), followed by Peptococci (13.7%), Proteus sp (9.4%), Enterococcus

sp (7.7%), S. aureus (7.7%), Clostridium sp (7.7%) and E coli (7.0%) [Louie et al., 1976].

Fieres et al., [1979], also reported a polymicrobial nature of infection in 56.6% patients

while 20% had S. aureus infection alone.

Sapico et al., (1980) did the qualitative assessment of 13 DFU patients in California, USA

and found that 84.6% patients had infection in their foot. A total of 4.7 strains per

patient were isolated (range 3-8). They have collected different samples (ulcer swab

pre/post amputed, curettage, saline aspirates and deep tissue biopsy) from the same

patient. Almost equal number of aerobic (51.7%) and anaerobic (43.1%) bacteria were

isolated and only 5.1% were fungal pathogens. Among the aerobic bacteria, majority

(53.3%) of infection in their foot were caused by Gram negative bacilli, Gram negative

cocci (3.3%) Gram positive cocci (36.6%) and Gram positive bacilli (6.6%). Among the

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anaerobic bacterial infections, majority (52%) were caused by gram positive bacilli

followed by Gram negative bacilli (44%) and gram positive cocci (4%).

Later in year 1984, Sapico and co-workers reconfirmed the polymicrobial nature in

their DFU patients. On an average, 4.8 organisms per patient were isolated. The high

prevalence of Bacteroides sp, Clostridium sp, gram negative enteric bacilli, anaerobic

bacteria and Enterococci were recovered in their study patients.

In the year 1996, another study by Tan et al., in Ohio, a total of 112 DFU patients were

studied and ulcer infection was found in 74.1% cases. Single isolates were found in

21.1%, and multiple organisms in 75.2%. Staphylococcus sp were the most common

single organisms isolated. Corynebacterium isolates were usually found as one of

multiple organisms. Corynebacterium and Streptococcus were recovered more

frequently from the wound and bone than were aerobic gram-negative rods.

Lipsky et al., [1997] studied the microbiology of DFU in Washington USA. 108 patients

were enrolled at 12 centres across the United States. Culture specimens were obtained

by swabbing in 72% of cases, by needle aspiration in 9%, and by tissue sampling in

19%. The mean number of pathogens isolated from culture of wound specimens taken

at the time of enrolment of the evaluable patients was 1.6 (range, 0-7). 92% of the

pathogens were aerobic organisms among them 67% were gram-positive cocci. In their

study, superficial swab and tissue specimens and needle aspirate gave almost same

results.

Amalia et al. [2002], studied microbiology of DFU in Manila. 19.4% patients of DFU had

no infection while 55% presented with monomicrobial infections and the remaining

suffered from polymicrobial infection. The most commonly isolated organism among all

patients was Proteus mirabilis (13%), followed by Enterococcus sp and E. coli (12% each

respectively)Among the anaerobic isolates, 26.7% were Peptostreptococcus sp, followed

by Actinomyces israelis (13.3%), Bifidobacterium sp and Clostridium sp (10.0% each).

A study by Abdulrazak et al., [2005] on 86 DFU patients from Kuwait showed an average

of 1.6 organisms per infection. 82.5% of the pathogens were aerobic organisms, among

them 73.6% were aerobic gram-positive cocci and 26.4% were gram-negative aerobes.

The most commonly isolated aerobic bacterial species were Staphylococcus aureus

(38.4%), Pseudomonus aeruginosa (l7.5%), and Protues mirabilis (l4%). Anaerobic

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infection were present in 10.5% Among anaerobic infection (10.5%), the most

frequently isolated bacteria were Bacteriodes fragilis and the fungal infection was

represented by 7%.

Another study conducted in Mahaboudha, Kathmandu by Sharma et al., [2006] reported

polymicrobial nature in 62.7% patients while 37.2% had monomicrobial infection.

Aerobic gram-positive cocci represented 73.6% of the isolates and gram-negative bacilli

in 36.4%. S. aureus was the most predominant (38.4%) isolate followed by P. aeruginosa

(17.5%) and Proteus mirabilis (14%).

Ako Nai et al., [2006] characterized the bacteria isolated from DFU infection in Nigeria.

All the patients studied were having infection. Among the bacterial isolates, 50.6% were

from superficial swabs and 49.3% from deep tissue biopsies. Altogether, 90.7% were

aerobes whereas 9.2% were strict anaerobes. Among aerobic Gram-positive cocci,

47.6% were S. aureus, coagulase negative staphylococci (33%) and Streptococcus spp.

(19.0%). However, among aerobic Gram-negative rods, Proteus species 32.9%, E. coli

26.1%, P. aeruginosa 12.5%, Klebsiella sp. 11.3%, Enterobacter cloaca 5.6%, Citrobacter

freundii (6.8%), while Serratia sp and Alcaligenes spp, contributed 3.4% and 1.1%,

respectively. 9.2% were strict anaerobes of which Corynebacterium spp. accounted for

16.0% of Gram-positive isolates.

Citron et al., (2007), in a multicentric trial (the SIDESTEP trial) conducted in United

States of America on 433 moderate to severely infected DFU patients isolated a total of

1,607 organisms from 454 specimens. Infections were present in 93.7% patients in

which 83.8% had polymicrobial infection and 16.2% had monomicrobial infection.

Gram-positive comprised 80.3% of the aerobic organisms. The predominant aerobic

species were S. aureus (76.6%). Enterococci were found in 35.7% of the patients.

Among Gram-negative rods, 19.7% were aerobic organisms. P. aeruginosa was the

predominant species followed by Proteus mirabilis and Klebsiella sp. Members of family

Enterobacteriaceae were the largest group comprised of 63.3% of all gram-negative

species. Anaerobes in 49% of patients, with gram-positive cocci accounting for 45.5%.

Finegoldia magna was the predominant anaerobic species in 24.4% patients. Prevotella

sp were the second most common (12.3%), followed by Porphyromonas sp (10.3%) and

the Bacteroides fragilis group (10.2%).

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In France, Sotto et al., [2007] studied 173 DFU patients. Polymicrobial infection was

found in 73.9% patients with 24.8% cases having monomicrobial infection. Aerobic

Gram-positive cocci were predominant (59.7%) with S. aureus (38.1%). Gram-negative

aerobic bacilli accounted for one-third of all microorganisms (31.7%) and among them,

organisms of Enterobacteriaceae were the most frequent pathogens. Of the anaerobes,

Peptostreptococcus sp. and Bacteroides sp. were the most commonly isolated species

(7.3% of all isolates).

Alavi SM et al., [2007] studied the relative frequency of bacterial isolates cultured from

32 diabetic foot ulcer patients in Ahwaz University, Iran. Infection was present in 81.2%

cases studied, in which polymicrobial infection was found in 50% of the cases and

monomicrobial infection in 31.2% cases. Aerobic Gram-positive bacteria accounted for

42.9 % and 54.8% were Gram-negative rods. S. aureus (26.2%) was the most frequent

microorganism, followed by E. coli (23.8%), P. mirabilis (9.5%), P. aeruginosa,

Enterobacter spp and Morganella spp (4.76% each). No anaerobes were isolated in their

study by using standard anaerobic culture methods.

In Malaysia, Raja [2007] from Malaysia, recovered 287 microbes from 194 DFU patients,

on an average of 1.47 organisms per patients. Polymicrobial growth was found in 42.7%

patients while 57.2% patients had pure growth. 52% were aerobic gram negative

bacteria while 45% showed gram positive aerobic bacterial infection. The organisms

isolated were S aureus (17%) followed by Proteus sp (15%), P aeruginosa (13%), Group

B Streptococcus (11%) and Bacteroides sp (1%). 1.7% anaerobes were isolated,

Peptotreptococcus spp, Bacteroides spp and Clostridium sp. Candida sp (0.69%) were

also isolated.

Lily SY et al., [2008] in their study from Singapore in DFU patients cultured 79% of total

samples received, which comprised of tissue (65.7%), swabs (28.9%) and bone (5.2%).

The most common anaerobic isolates were Peptostreptococcus sp (47%) and

Bacteroides fragilis group (19%), Prevotella sp (3%), Clostridium sp (2%), Fusobacterium

sp (2%), Eggerthella sp and Acidominococcus sp (1% each respectively).

Tascini et al., [2011], studied the microbiology of moderate to severe diabetic foot

infections from Pisa, Italy. In their study, a total of 4332 samples were collected from

1295 patients with an average of 3.3 samples per patient, over three years. Among 1295

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specimens; 45.1% were swabs, 43.6% were aspirated samples and 11.1% were tissue

specimens. Specimens collected by aspiration yielded a positive culture more frequently

than swabs and tissue specimens (72.4% vs 59.7% and 50.3%). About 40% of the

positive samples were polymicrobial. The Gram positive bacteria were more frequently

isolated (52.6%) and S. aureus was the most commonly isolated organism (29.9%).

Enterococcus spp. was isolated in around 10% of samples, mainly E. faecalis.

Streptococci were only 4.6% of isolates. The Gram negative rods were isolated from

40.6% of cases, consisting of Enterobacteriaceae (23.5%) and P. aeruginosa (10.3%).

Anaerobes were isolated in only 0.3% of cases. Candida sp was isolated from 6% of

cases.

2.15.2. Indian Scenario.

Anandi et al., [2004] studied the bacterial etiology of 107 diabetic foot ulcer patients

from India. Infection was present in 84.1% cases, in which polymicrobial etiology was

observed in 76.6% patients and only 23.3% had monomicrobial infection. Aerobic

bacteria were isolated from 79.2% while 20.2% showed anaerobic infection. The most

common aerobic bacteria was E. coli (27.7%) followed by Proteus sp (16.9%), S. aureus

and Klebsiella sp (13.6% each). The commonest among anaerobes was Clostridium spp

(60%), followed by Bacteroides fragilis (20%), Prevotella sp (13.3%) and

Peptostreptococcus sp (6.7%).

In another Clinico-microbiological study of diabetic patients by Gadepalli et al., [2006]

in North India on 80 cases having infection in their foot. Isolated a total of 183

organisms on an average of 2.3 species per patients. Polymicrobial infection was found

in 82.5% patients and remaining was monomicrobial infection. The majority (65.0%)

were infected with aerobes only while anaerobic infection alone was found in 1.2%

patients. Both aerobic and anaerobic organisms’ infection was found in 33.8% patients.

Gram-positive organisms were found in 13.8% patients, and 28.7% patients had gram-

negative organisms. The remaining 57.5% had both Gram-positive and Gram-negative

organisms.

Bansal et al., [2008] from Chandigarh, India studied 103 DFU patients. Infection was

present in 93.2% cases in which polymicrobial infection was present in 37.5% patients

and 62.5% had monomicrobial infection. A total of 157 organisms (91.2% aerobic, 8.9%

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fungi) were isolated, averaging of 1.52 organisms per patient. In 85.6% specimens, only

bacteria were isolated and, mixed infection of bacteria and fungi in 8.4% specimens. In

their study, they reported that gram negative and gram positive organisms were 76%

and 24% respectively. P aeruginosa (21.6%) were the most common organisms

isolatedfollowed by S aureus (18.8%), E coli (18.1%) and K pneumonia (16.7%). Fungal

isolates comprises of 9% of total samples. Candida tropicalis (29%), C albicans (14%),

and C guililermondi (7%); followed by Aspergillus flavis (21%), A niger (14%) and

Fusarium (14%).

Recently, Ramakant et al., (2011) studied the changing microbiological profile of

pathogenic bacteria isolated from DFU in SGPGI, Lucknow, India over a period of 8

years. 1632 cultures were isolated from 434 DFU patients, showing polymicrobial

infection in 66% cases while 23% had monomicrobial infection. In their study, they

reported that Gram-negative bacterial infection was increasing from 50.6% to 66%. The

most common isolate was P. aeruginosa (16.9%) followed by E. coli (16.1%) and Proteus

spp. (8.8%). Other Gram negative aerobes recovered were Citrobacter sp., Enterobacter

spp. and Acinetobacter spp.

Umadevi et al., [2011] conducted a prospective study on DFU infections for more than

one year in Pondicherry, India. A total of 171 bacteria were isolated from 105 DFU

patients. Infection was present in 89.7% case, in which 44.8% had monomicrobial

infection and 52.4% had polymicrobial infection. Gram positive organisms infection

only was found in 8.6% patients, 52.4% had only Gram negative organisms infection

while remaining 39.0% had mixed gram positive and gram negative. The most

commonly found bacteria was Klebsiella pneumonia (20.5%) followed by Pseudomonas

aeruginosa and S aureus (17.0% each), E coli (14.6%), CONS (7.0%), Proteus mirabilis

(5.8%), Enterococcus sp (5.3%), Citrobacter sp (4.1%), Proteus vulgaris (3.5%)

Acinetobacter sp (3.5%), Pseudomonas sp (1.2%) and Providencia sp (0.6%).

Recently, Tiwari et al., 2012 conducted a prospective study from BHU, Varanasi, India.,

on 62 cases, 43.5% had mono-microbial infection, 35.5% had poly-microbial infections,

and 21% had sterile culture. A total of 82 bacteria were isolated, 68% were Gram

negative and 32% were Gram positive. E. coli was the most common pathogen isolated

followed by S. aureus. Other commonly isolated bacteria were P. aeruginosa,

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Streptococci, P. mirabilis, Citrobacter sp., P. vulgaris, K. pneumoniae, Bacillus sp.,

Morganella sp., Acinetobacter sp., E. faecalis, K. oxytoca, E. aerogenes, Coagulase –ve

Staph, Pneumococcus, Enterococci. Co-infection with Candida spp. was also found in one

case with Gram-negative infection (E. coli).

2.16. Antibiotic Resistance in Diabetic Foot Ulcer.

The infection of DFU and the drug resistance in this group of patients is an important

and major health issue which needs to be addressed. The problem of increasing

population who is at risk of developing diabetes and subsequently ulcer, along with the

increasing prevalence of antibiotic resistance, makes this a highly pertinent issue.

Moreover, the polymicrobial nature is likely to provide an appropriate environment for

genetic exchange between bacteria making these problems worse.

Different populations of DFU patients show wide variation in the level of antibiotic

resistance encountered. A prospective study of uninfected chronic venous leg ulcer from

66 patients who had received no antibiotics in the previous month showed a very low

level of antibiotic resistance, only two patients were found to have MRSA (Davies,

2002). Day & Armstrong (1997) reviewed the limited evidence on risk factors for the

carriage of resistance in diabetic foot wounds. While they found no studies that had

directly addressed this issue, suggested risks include cross-contamination of wounds

from the patients themselves, inanimate objects or health care personnel, long-term use

of antibiotics, prior hospitalization and severity of illness. High prevalence of antibiotic

resistance, especially MRSA, affects treatment decisions concerning wounds and raises

the question of whether the empirical regimens could cover these resistant organisms

(Armstrong et al., 2004). Whilst the additional impact of antibiotic-resistant organisms

on wound healing is not known, overall, the morbidity, mortality and cost associated

with infections in hospitalised patients caused by antibiotic-resistant organisms has

been shown to be 1.3- to 2-fold higher than those infections caused by antibiotic-

sensitive organisms (Cosgrove and Carmeli, 2003).

2.16.1. Global Scenario.

Abdulrazak et al., [2004] studied the diabetic foot infections in Kuwait. A total of 140

bacteria were isolated from 86 patients and antimicrobial susceptibility showed that

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imipenem, meropenem, and cefepime were the most effective agents against all the

isolated gram negative bacterial species. The susceptibility exhibited by all the bacterial

isolates against ciprofloxacin, ceftazidime, cefotaxime, gentamycin, and amikacin was

between 80-100%. The workers showed that the most effective and promising agents

are imipenem, meropenem, piperacillin and clindamycin. Metronidazole was found

effective for anaerobes. Candida isolates were susceptible to Amphotericin B, nystatin,

econazole and fluconazole. They suggested that the spectrum of causative organisms,

their resistant patterns, efficacy, and safety should be taken into account before

choosing antimicrobials for the treatment.

Earlier, Amalia et al., [2002] reported the sensitivity of anaerobic bacteria isolated from

DFU patients. All isolates were tested to metronidazole, clindamycin, cefoxitin,

imipenem, penicillin, and ampicillin-sulbactam using the E-test method. Of the total

isolates tested, 48% were resistant to metronidazole and 24% were resistant to

clindamycin. Imipenem and ampicillin-sulbactam had the lowest resistance rates (3.4%

each). For both gram-positive cocci and bacilli, clindamycin also had high resistance

rates at 22% and 27% respectively. 40% gram-positive bacilli, were resistant to

metronidazole while 20% were resistant to penicillin. None of the gram-negative bacilli

isolated showed resistance to imipenem or to ampicillin-sulbactam. Anaerobes were

found resistant to metronidazole but sensitive to all the other antibiotics tested.

Ako-Nai et al., [2006] characterized 152 DFU isolates from Nigeria. Sensitivity testing

was done against 10 different antibiotic grouped as penicillins, tetracyclines,

macrolides, nitrofurans, chloramphenicol, trimethoprim, aminoglycosides and

quinolones. Sensitivity tests carried out with 70 different isolates recovered from

superficial swabs revealed amoxicillin and cloxacillin to show the highest values of

resistance, 64.3% and 71.4%, respectively. Resistance to erythromycin was 67.1% and

tetracycline 61.4%. Similarly, 48.6% of the isolates were resistant to cotrimoxazole

while resistance to chloramphenicol was 45.7%. Resistance to a relatively new beta-

lactamase-resistant antibiotic, augmentin was seen in 38.6% of the isolates. The results

of resistance profile of isolates recovered from deep tissue aspirates showed resistance

to amoxicillin as highest (86.8%) however, 77.9% to tetracycline. Only 7.4% of these

isolates were resistant to ofloxacin. Among the anaerobes screened, 55% were resistant

to gentamicin while 42.9% were resistant to cotrimoxazole.

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Alavi et al., [2007], reported the sensitivity pattern of 42 bacterial isolates from 32 DFU

patients. Antibacterial susceptibility testing revealed that, S aureus isolates were

resistant to all the tested antibiotics except for Ciprofloxacin and Amikacin for which

the sensitivity rates were 91% and 80% respectively. All the gram negative isolates

were resistant to cloxacillin, amoxycillin, clindamycin and vancomycin except that 50%

of isolates of Proteus mirabilis were susceptible to these antibiotics. Pseudomonas

aeruginosa was the second most resistant isolate with resistance to all antibiotics used

and have 100% sensitivity to Ciprofloxacin and 50% to Ceftriaxone. They concluded

that the rate of resistance was 65% among the bacterial isolates.

Raja et al., [2007] reported the resistance to methicillin in S aureus isolates as 16%,

while it was sensitive to vancomycin and rifampin. Resistance to fusidic acid was found

in 7% cases. Sensitivity to penicillin, ampicillin, vancomycin, imipenem, cefuroxime and

clindamycin by group B streptococci was found as 100%. Enterococcus sp were detected

resistant to imipenem (8%), ampicillin (17%) and co-trimoxazoles (25%). In their

study, least resistance was shown by Gram negative bacilli to imipenem and amikacin

while ciprofloxacin, piperacillin tazobactam, amoxicillin clavulanic acid and ampicillin

sulbactam showed satisfactory activity. Metronidazole, imipenem and clindamycin had

good activity against all anaerobes. Imipenem was effective against gram negative and

gram positive isolates equally in their study.

Citron et al., [2007] studied 454 DFU patients and isolated 1607 organisms. All aerobic

gram-positive organisms were susceptible to vancomycin, daptomycin, and linezolid.

Piperacillin-tazobactam and amoxicillin-clavulanate were the next most active drugs

against the gram-positive aerobes. Ciprofloxacin was the least sensitive throughout of

the quinolones, especially against all species of Streptococci, however, moxifloxacin was

the most active quinolone. Among the gram-negative organisms, Stenotrophomonas

maltophilia was resistant to most agents tested. P aeruginosa strains and the

Enterobacteriaceae group were largely susceptible to imipenem, piperacillin-

tazobactam, ceftazidime, aminoglycosides, and ciprofloxacin. Piperacillin tazobactam

and the quinolones were active against more than 90% of the gram-negative organisms,

while amoxicillin-clavulanate, doxycycline, and cephalexin were the least active of the

drugs tested. Among the anaerobes, all isolates were susceptible to ertapenem except

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two strains, one B. fragilis strain and one Bacteroides vulgates. Overall, 18% of the

anaerobes were resistant to clindamycin and 24% were resistant to moxifloxacin.

Lily SY et al., [2008] out of 99 anaerobic bacteria reported that 81% were susceptible to

clindamycin, 98% to imipenem and 99% to metronidazole. Clindamycin resistance was

predominantly present in the Bacteroides fragilis group and Peptostreptococci, while

imipenem resistance was present in 2 Fusobacterium spp. Metronidazole resistance was

present in 1 Peptostreptoccus sp. isolate. The results of this study showed that

metronidazole and imipenem resistance remained low.

Tascini C et al., 2011 studied the antimicrobial resistance in DFU isolates. All S. aureus

isolates were susceptible to vancomycin and teicoplanin and 22% were found MRSA

positive. 85% were susceptible to Co-trimoxazole, doxycicline and rifampin. Piperacillin

tazobactam, cefepime, ceftazidime and meropenem were active against 80% of P

aeruginosa. Imipenem was active against 74% of strains. Quinolones susceptibility was

around 60% with ciprofloxacin, more active than levofloxacin. Levofloxacin had better

clinical activity as compared to other quinolones. All the 70 Candida isolates were

susceptible to fluconazole except one strain of Candida ciferrii.

2.16.2. Indian Scenario.

Anandi et al., [2004] studied the antibiotic susceptibility of diabetic foot isolates from

107 patients. The average sensitivity of E coli was 97% followed by Klebsiella sp (94%),

Proteus spp (92%), Enterobacter sp (90%) and Pseudomonas sp (84%) for ciprofloxacin,

ofloxacin and pefloxacin. All the aerobic isolates were sensitive to amikacin and

gentamycin and, cefotaxime in their study. They concluded that the results of sensitivity

testing played an important role in the treatment of infection in patients especially with

cellulitis and gangrene.

In a study conducted by Sharma et al., [2006] on 43 patients. In 82.2% of cases S aureus

were resistant to ampicillin. The most sensitive drug was found to be gentamicin.

Cloxacillin and Ciprofloxacin were second most sensitive drug, having a resistance of

11.6% for S aureus. No resistance was found in streptococcus isolates against Cloxacillin

and Ciprofloxacin. E coli were resistant to almost all the drugs studied. Amikacin had a

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slightly better sensitivity for E coli. Amikacin remained the best antibiotic for Proteus

and Pseudomonas also.

Gadepalli et al., [2006] studied the antibiotic resistance of 183 bacterias isolates from

80 DFU patients. The S aureus exhibited a high frequency (56.0%) of resistance to the

antibiotics tested. High levels of resistance to erythromycin, tetracycline, and

ciprofloxacin (40% each) were found in Enterococcus species. High-level of

aminoglycoside resistance was not observed in the Enterococcal isolates. All the isolates

were uniformly susceptible to vancomycin and linezolid. All the anaerobes were

susceptible to metronidazole and amoxicillin clavulanate.

Bansal et al., [2008] isolated 157 organisms from 103 DFU patients and studied their

sensitivity patter. 55.5% were MRSA and almost all the isolates were sensitive to

ceftriaxone and imipenem. Amikacin and ciprofloxacin shows good sensitivity. E fecalis

showed 75% resistance to erythromycin and 42% to amoxycillin. All the isolates were

85% sensitive to cefoparazone sulbactam, 71% for amoxycillin clavulanic acid, 62% for

ciprofloxacin, and 66% for gentamycin. P aeruginosa were sensitive to cefoparazone

sulbactam, ceftazidime and imipenem. E coli showed 100% sensitivity to imipenem.

Ramakant et al., [2011] showed antibiotic sensitivity patterns change before and after

1999: piperacillin–tazobactam 74% vs 66%, imipenem 77% vs 85%, cefoperazone–

sulbactam 47% vs 44%, amikacin 62% vs 78%, ceftriaxone 41% vs 36%, amoxicillin–

clavulanate 51% vs 43% and clindamycin 43% vs 36% respectively. They suggested

that the treatment modes can be modified based on the severity of infection and on any

available microbiological data such as recent culture results or current Gram-stained

smear findings.

Umadevi et al., [2011] demonstrated that 65.5% of S aureus were MRSA positive. 56% of

Enterobacteriaceae member were ESBL producers, in which 62.5% of Proteus spp were

ESBL positive followed by Klebsiella pneumoniae (60%) and Escherichia coli (56%).

Twenty two multi-drug resistant gram-negative bacteria were also observed. Amikacin,

piperacillin tazobactam, imipenem were sensitive against gram-negative bacilli, while

vancomycin was sensitive against gram-positive bacteria.

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Murugan et al., 2011 studied antimicrobial susceptibility pattern of ESBL producing E

coli isolated from DFU. E coli exhibited 100% susceptibility to imipenem and

meropenem and resistant to cephalexin, erythromycin, gentamycin and norfloxacin.

Intermediate resistance was 76.5% against cefotaxime. ESBL positive isolates showed

52.9% resistance to amikacin, 64.7% to gentamicin, 70.6% to cloxacin, 76.5% to co-

trimoxazole and 82.4% to cephalexin.

It is clear from the literature that antibiotics have an important role to play in the

treatment of clinically infected diabetic foot. However, there are no conclusive scientific

studies to support antibiotic use that might definitively guide antibiotic choice, dose and

duration. The use of antibiotics is not risk-free for the individual with both the

immediate risk associated with anaphylactic reactions (Wilson, 2000) and the longer

term prospect of antibiotic use making co-morbidities more difficult to treat. In

addition, antibiotic resistance in the general population is a continuing and growing

concern. The contribution made to the development, maintenance and dissemination of

resistance by those antibiotics issued for DFUs is not yet known, although there is

reason to believe that the DFU patient population may be of importance due to the high

levels of antibiotic prescribing to these patients, the degree of microbial load associated

with their lesions and the potential they provide for dissemination of resistant

organisms to others. The resistant organisms have been isolated from both infected and

colonized chronic wounds, however, the true prevalence and impact on the wider

community are, again, not known. Research needs to be undertaken to elicit the

interactions between microbes, antibiotics and antibiotic resistance in chronic wounds

for the benefit of both chronic wound patients and the population in general.

2.17. Extended-Spectrum Beta Lactamases (ESBL).

ESBLs are a group of enzymes that break down antibiotics belonging to the penicillin

and cephalosporin groups and render them ineffective. ESBL has traditionally been

defined as transmissible beta-lactamases that can be inhibited by clavulanic acid,

tazobactam or sulbactam, and which are encoded by genes that can be exchanged

between bacteria.

There is no consensus of the precise definition of ESBLs. A commonly used working

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definition is that the ESBLs are beta lactamases capable of conferring bacterial

resistance to the penicillins, first, second, and third-generation cephalosporins,

and aztreonam (but not the cephamycins or carbapenems) by hydrolysis of these

antibiotics, and which are inhibited by betalactamase inhibitors such as

clavulanic acid.

2.17.1. History, Evolution and Dissemination of Β-Lactamases.

The increase in antibiotic resistance among Gram-negative bacteria is a notable

example of how bacteria can procure, maintain, and express new genetic information

that can confer resistance to one or several antibiotics. This genetic plasticity can occur

both inter- and intragenerically. Gram-negative bacterial resistance possibly now equals

to gram-positive bacterial resistance and has prompted calls for similar infection

control measures to curb their dissemination. Reports of resistance vary, but a general

consensus appears to prevail that quinolone and broad-spectrum β-lactam resistance is

increasing in members of the family Enterobacteriaceae and Acinetobacter spp. and that

treatment regimens for the eradication of Pseudomonas aeruginosa infections are

becoming increasingly limited.

The introduction of the third-generation cephalosporins into clinical practice in the

early 1980s was heralded as a major breakthrough in the fight against beta-

lactamase-mediated bacterial resistance to antibiotics. These cephalosporins had

been developed in response to the increased prevalence of beta lactamases in

certain organisms (for example, ampicillin hydrolyzing TEM-1 and SHV-1 beta-

lactamases in Escherichia coli and Klebsiella sp). Not only were the third

generation cephalosporins effective a g a i n s t m o s t beta lactamase-producing

organisms but they had the major advantage of lessened nephrotoxic effects

compared to aminoglycosides and polymyxins.

The first report of plasmid-encoded beta-lactamases capable of hydrolyzing the

extended-spectrum cephalosporins was published in 1983 (Knothe et al., 1983).

The gene encoding the b e t a - lactamase showed a mutation of a single

nucelotide compared to the gene encoding SHV-1. Other beta-lactamases were

soon discovered which were closely related to TEM-1 and TEM-2, but had the

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ability to confer resistance to the extended-spectrum cephalosporins (Sirot et al.,

1987, Brun-Buisson et al., 1987). Hence these new beta-lactamases were coined

extended-spectrum beta-lactamases (ESBLs). In the first substantial review of

ESBLs in 1989, it was noted by Philippon, Labia, and Jacoby that the ESBLs

represented the first example in which beta lactamase-mediated resistance to

beta-lactam antibiotics resulted from fundamental changes in the substrate

spectra of the enzymes (Philippon et al., 1989).

Gram-negative bacteria have at their disposal a plethora of resistance mechanisms that

they can sequester and/or evince, eluding the actions of carbapenems and other β-

lactams. The common form of resistance is either through lack of drug penetration (i.e.,

outer membrane protein [OMP] mutations and efflux pumps), hyper production of an

AmpC-type β-lactamase, and/or carbapenem-hydrolyzing β-Iactamases. Based on

molecular studies, two types of carbapenem-hydrolyzing enzymes have been described:

serine enzymes possessing a serine moiety at the active site, and metallo β-lactamases

(MBLs), requiring divalent cations, usually zinc, as metal cofactors for enzyme activity

(Bush et al., 1999).

The series Carbapenems are invariably derivatives of class A or class D enzymes and

usually mediate Carbapenem resistance in Enterobacteriaceae or Acinetobacter spa The

enzymes characterized from Enterobacteriaceae include NmcA, Smel-3, IMI-1, KPC1-3,

and. GES-2, Despite the avidity of these enzymes for carbapenems, they do not always

mediate high-level resistance and not all are inhibited by clavulanic acid. In contrast, the

oxacillinases have been characterized from Acinetobacter brumannii only and include

OXA 23 to 27, OXA-40, and OXA-48. These enzymes hydrolyze carbapenems poorly but

are able to confer resistance and are only partially inhibited by clavulanic acid. The class

A and class D carbapenemases are encoded by genes that have been procured by the

bacterium and can be chromosomally encoded (sometime associated with integrons) or

carried on plasmids.

Extended-spectrum p-lactamases (ESBLs) and AmpC β-iactamases are of increasing

clinical concern. ESBLs are most commonly produced by Klebsiella spp. and Escherichia

coii but may also occur in other gram-negative bacteria. They are typically plasmid

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mediated, ciavulanate susceptible enzymes that hydroiyze penicillins, expanded-

spectrurn cephalosporins (cefotaxime, ceftriaxone, ceftazidime, cefepirne and others)

and aztreonam. AmpC class p-lactamases are cephalosporinases that are poorly

inhibited by clavulanic acid. They can be differentiated from other ESBLs by their ability

to hydrolyze cephamycins as well a3 other extended-spectrum cephalosporins. AmpC p-

lactamases, demonstrated or presumed to be chromosomaily o;- plasmid mediated,

have been described in pathogens e.g., Klebsiella pneunioniae, Escherichia coli,

Salmonella spp., Proteus mirabilis, Citrobacter freundii, Acinetobacter, Enterobacter spp

and. Pseudomonas aeruginosa. Although reported with increasing frequency, the true

rate of occurrence of AmpC β-iactamases in different organisms, including members of

Enterobacteriaceae, remains unknown.

In 2001, the ESBLs were reviewed by Patricia Bradford (Bradford, 2001). The body of

knowledge pertaining to ESBLs has grown rapidly since that time. A Pub-Med

search using the key-words extended-spectrum β-lactamase reveals more than

1,300 relevant articles, with more than 600 published since the time Bradford’s

review was written.

The total number of ESBLs now characterized exceeds to 200. These are detailed

o n the website of the nomenclature of ESBLs hosted by George Jacoby and Karen

Bush (http://www.lahey.org/studies/webt.htm). Published research on ESBLs

has now originated from more than 30 different countries, reflecting the truly

worldwide distribution of ESBL producing organisms.

However, primarily due to genomic sequencing, increasingly more chromosomally

mediated genes are being discovered but are often found in obscure nonclinical

bacteria.

2.17.2. Mechanism of action of β lactamases.

β-Lactamase enzymes destroy the β -lactam ring by two major mechanisms of action.

Firstly, most common β -lactamase have a serine based mechanism of action. They are

divided in to three major classes (A, C and D) on the basis of the ammo acid sequences.

They contain an active site consisting of a narrow longitudinal groove, with a cavity on

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its floor (the oxyanion pocket), which is loosely constructed in order to have

conformational flexibility in terms of substrate binding. Close to this lies, the serine

residue that irreversibly reacts with the carbonyl carbon of the β -lactam ring, resulting-

in an open ring (inactive β -lactam) and regenerating the β-lactamase. These enzymes

are active against many penicillins, cephalosporins and monobactam. Secondly a less

commonly encountered group of β -lactamases is the metallo β-lactamases or class B

β-lactamases. These use a divalent transition metal ion most often zinc, linked to a

histidine or cysteine residue or both, to react with the carbonyl group of the amide bond

of most penicillins, cephalosporins and carbapcnems but not monobactams (Samaha-

Kfoury et al, 2003).

2.17.3. Classification of β – lactamases.

Classifications involve two major approaches, first and older one based on the

biochemical and functional characteristics of the enzyme and the second approach is

based on molecular structure of enzyme.

Functional classification schemes were started by Sawai et al. in 1968 describing

penicillinases and cephalosporinases by using the response to antisera -as an additional

discriminator. Jack and Richmond in 1970 proposed a classification scheme and was

expanded by Richmond and Sykes in 1973. Their classification was based on hydrolytic

spectrum, substrate profile and whether they are encoded by chromosome or by

plasmids. Sykes and Mathew in 1976 emphasized the plasmid mediated beta -

lactamases could be differentiated by isoelectric focusing. Mitsuhashi and Inoue in 1981

added the category "cefuroxime hydrolyzing beta - lactamase" to the "penicillinase and

cephalosporinase". Bush in 1989 expanded the substrate profile, added the reaction

with EDTA, and correlated between function and molecular classification.

Molecular structure classifications were first proposed by Ambler in 1980 when only

four ammo acid sequences of β -lactamases were known. At that time a single class of

serine enzyme was designated, the class A β-lactamases that included the

Staphylococcus aureus PCI penillinase, in contrast to the class B metallo β-lactamases

from Bacillus cereus. The class C cephalosporinases were discovered by Jaurin and

Grundstrom in 1981, and class D oxacillinases were segregated from other serine p-

lactamases in late 1980s (Ouellette et al. 1987 & Huovinen et al. 1988). However, in

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1995 Bush, Jacoby and Medeiros gave the latest classification of β -Iactamases based on

biochemical properties, molecular structure and amino acid sequence. They suggested

classification in to four groups (1-4) on the basis of the spectrum of activity and other

functional characteristics.

2.17.3.1. Schematic outline of Functional Classification β-lactamases. [Bush et al.,

1995].

GROUP 1: Cephalosporinase, Molecular Class C (not inhibited by clavulanic acid).

They are cephalosporinases not inhibited by clavulanic acid, belonging to the molecular

class C.

GROUP 2: Penicillinases, Cephalosporinases, or both inhibited by clavulanic acid,

corresponding to the molecular classes A and D reflecting the original TEM and SHV

genes. However, because of the increasing number of TEM- and SHV-derived {beta}-

lactamases, they were divided into two subclasses, 2a and 2b.

Group 2a: Penicillinase, Molecular Class A

The 2a subgroup contains just penicillinases.

Group 2b Broad-Spectrum, Molecular Class A

2b Opposite to 2a , 2b are broad-spectrum {beta}-lactamases, meaning that

they are capable of inactivating penicillins and cephalosporins at the same

rate. Furthermore, new subgroups were segregated from subgroup 2b.

GROUP 2be: Extended-Spectrum, Molecular Class A

Subgroup 2be, with the letter "e" for extended spectrum of activity,

represents the ESBLs, which are capable of inactivating third-generation

cephalosporins (ceftazidime, cefotaxime, and cefpodoxime) as well as

monobactams (aztreonam).

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GROUP 2br: Inhibitor-Resistant, Molecular Class A (diminished inhibition

by clavulanic acid)

The 2br enzymes, with the letter "r" denoting reduced binding to clavulanic

acid and sulbactam, are also called inhibitor-resistant TEM-derivative

enzymes; nevertheless, they are commonly still susceptible to tazobactam,

except where an amino acid replacement exists at position at 69.

GROUP 2c: Carbenicillinase, Molecular Class A

Later subgroup 2c was segregated from group 2 because these enzymes

inactivate carbenicillin more than benzylpenicillin, with some effect on

cloxacillin.

GROUP 2d: Cloxacilanase, Molecular Class D or A

Subgroup 2d enzymes inactivate cloxacillin more than benzylpenicillin, with

some activity against carbenicillin; these enzymes are poorly inhibited by

clavulanic acid, and some of them are ESBLs. The correct term is

"OXACILLINASE". These enzymes are able to inactivate the

oxazolylpenicillins like oxacilli, cloxacilli, dicloxacillin. The enzymes belong

to the molecular class D not molecular class A.

GROUP 2e: Cephalosporinase, Molecular Class A

Subgroup 2e enzymes are cephalosporinases that can also hydrolyse

monobactams, and they are inhibited by clavulanic acid.

GROUP 2f: Carbapenamase, Molecular Class A

Subgroup 2f was added because these are serine-based carbapenemases, in

contrast to the zinc-based carbapenemases included in group 3.

GROUP 3: Metalloenzyme, Molecular Class B (not inhibited by clavulanic acid).

Group 3 are the zinc-based or metallo {beta}-lactamases, corresponding to the

molecular class B, which are the only enzymes acting by the metal ion zinc, as discussed

above. Metallo B-lactamases are able to hydrolyse penicillins, cephalosporins, and

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72

carbapenems. Thus, carbapenems are inhibited by both group 2f (serine-based

mechanism) and group 3 (zinc-based mechanism)

GROUP 4: Penicillinase, No Molecular Class (not inhibited by clavulanic acid)

Group 4 are penicillinases that are not inhibited by clavulanic acid, and they do not yet

have a corresponding molecular class.

2.17.3.2. Molecular Classification of ESBL.

The majority of ESBLs contains a serine at the active site and belongs to Ambler's

molecular class A. Class A enzymes are characterized by an active-site serine, a

molecular mass of approximately 29,000 Da, and the preferential hydrolysis of

penicillins Class A β -lactamases include such enzymes as TEM-1, SHV-1, and the

penicillinase found in S. aureus. The molecular classification scheme is still used to

characterize β -lactamases; however, it does not sufficiently differentiate the many

different types of class A enzymes. The classification scheme was based on the substrate

profile and the location of the gene encoding the p-lactamase. This classification scheme

was developed before ESBLs arose, and it did not allow for the differentiation between

the original TEM and SHV enzymes and their ESBL derivatives. More recently, a

classification scheme was devised by Bush, Jacoby, and Medeiros (1995) that uses the

biochemical properties of the enzyme plus the molecular structure and nucleotide

sequence of the genes to place β-lactamases into functional groups. Using this scheme,

ESBLs are defined as β -lactamases capable of hydroiyzing oximino-cephalosporins that

are inhibited by clavuianic acid and are placed into functional group 2be (Bush K, et al.,

1995).

2.17.4. Diversity of ESBL Types.

2.17.4.1. SHV beta-Lactamases.

The SHV-type ESBLs was frequently found in clinical isolates than any other

type of ESBLs (Jacoby et al., 1997). SHV refers t o sulfhydryl variable. This

designation was made because it was thought that the inhibition of SHV activity

by p-chloromercuribenzoate was substrate related, and was variable according to

the substrate used for the assay (Sykes and Bush, 1982). (This activity was never

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confirmed in later studies with purified enzyme.) In 1983, a Klebsiella ozaenae

isolate from Germany was discovered which possessed a beta-lactamase which

efficiently hydrolyzed cefotaxime, and to a lesser extent ceftazidime (Knothe et. al.,

1983). Sequencing showed that the betalactamase differed from SHV-1, by

replacement of glycine by serine at the 238 position. This mutation alone accounts

for the extended-spectrum properties of this beta lactamase, designated SHV-2.

Within 15 years of the discovery of this enzyme, organisms harbouring SHV-2

were found in every inhabited continent (Paterson et al., 2003), implying that

selection pressure from third-generation cephalosporin in the first decade of

their use was responsible. SHV-type ESBLs have been detected in a wide range of

Enterobacteriaceae (Huang et. al., 2004; Poirel et. al., 2004)

2.17.4.2. TEM beta-Lactamases.

The TEM-type ESBLs are derivatives of TEM-1 and TEM-2. TEM-1 was first

reported in 1965 from an Escherichia coli isolate from a patient in Athens, Greece,

named Temoneira (hence the designation TEM) (Datta and Kontomichalou, 1965).

TEM-1 is able to hydrolyze ampicillin at a greater rate than carbenicillin, oxacillin, or

cephalothin, and has negligible activity against extended-spectrum cephalosporins.

It is inhibited by clavulanic acid. TEM-2 has the same hydrolytic profile as TEM-1,

but differs from TEM-1 by having a more active native promoter and by a difference

in isoelectric point (5.6 compared to 5.4). TEM-13 also has a similar hydrolytic

profile to TEM-1 a n d TE M -2 (Jacoby and Medeiros, 1991). TEM-1, TEM-2, and TEM-

13 are not ESBLs. However, in 1987 Klebsiella pneumoniae isolates detected in France

as early as 1984 were found to harbor a novel plasmid-mediated beta lactamase

coined CTX-1 (Sirot et al., 1987; Bush et al., 1995). The enzyme was originally named

C T X -1 because of its enhanced activity against cefotaxime. The enzyme, now

termed TEM-3, differed from TEM-2 by two amino acid substitutions (Sougakoff

et. al., 1988).

In retrospect, TEM-3 may not have been the first TEM-type ESBL. Klebsiella oxytoca,

harboring a plasmid carrying a gene encoding ceftazidime resistance, was first

isolated in Liverpool, England, in 1982 (Du Bois et. al., 1995). The responsible beta

lactamase was what is now called TEM-12. Interestingly, t h e strain came from a

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neonatal unit which had been stricken by an outbreak of Klebsiella oxytoca

producing TEM-1. Ceftazidime was used to treat infected patients, but subsequent

isolates of Klebsiella oxytoca from the same unit harboured the TEM-type ESBL (Du

Bois et. al., 1995). This is a good example of the emergence of ESBLs as a response to

the selective pressure induced by extended-spectrum cephalosporins. Well over

100 TEM-type beta lactamases have been described, of which the majority are ESBLs.

Their isoelectric points range from 5.2 to 6.5. The amino acid changes in comparison

w i t h TEM-1 o r TEM-2 are documented at http://www.lahey.org/ studies/

temtable.htm.

A number of TEM derivatives have been found which have reduced affinity for beta

lactamase inhibitors. These enzymes have been reviewed elsewhere (Chaibi et. al.,

1999). With very few exceptions, TEM-type enzymes which are less susceptible to

the effects of beta lactamase inhibitors have negligible hydrolytic activity against the

extended-spectrum cephalosporin and are not considered ESBLs. However,

interesting mutants of TEM beta lactamases are being recovered that maintain the

ability to hydrolyse third generation cephalosporins but also demonstrate inhibitor

resistance. These are referred to as complex mutants of TEM (Fiett et. al., 2000;

Neuwirth et. al., 2001; Poirel et. al., 2004b).

2.17.4.3. CTX-M beta-Lactamases.

The name CTX reflects the potent hydrolytic activity of these beta lactamases

against cefotaxime. Organisms producing CTX-M-type beta lactamases typically

have cefotaxime MICs in the resistant range (>64 μg/ml), while ceftazidime MICs

are usually in the apparently susceptible range (2 to 8 μg/ml). However, some

CTX-M-type ESBLs may actually hydrolyse ceftazidime and confer resistance to

this cephalosporin (MICs as high as 256 μg/ml) (Baraniak et. al., 2002; Poirel et. al.,

2002; Sturenburg et. al., 2004). Tazobactam exhibits an almost 10-fold greater

inhibitory activity than clavulanic acid against CTX-M-type beta lactamases

(Bush et. al., 1993). It should be noted that the same organism may harbor both

CTX-M-type and SHV-type ESBLs or CTX-M-type ESBLs and AmpC-type beta

lactamases, which may alter the antibiotic resistance phenotype (Yan et. al., 2000).

The number of CTX-M-type E S B L s is rapidly expanding. They have now been

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detected in every populated continent (Baraniak et. al., 2002; Radice et. al., 2002;

Bou et. al., 2002; Ma et. al., 2002; Dutour et. al., 2002; Cao et. al., 2002; Alobwede et. al.,

2003, Brenwald et. al., 2003; Paterson et. al., 2003; Moland et. al., 2003; Chanawong et.

al., 2002). In recent years, a number of authors have reported the advent of CTX-

M-type ESBLs in these d i f f e r e n t regions (Moland et. al., 2002; Alobwede et. al.,

2003; Bonnet, 2004; Moland et. al., 2003; Pitout et. al., 2004). Given the widespread

findings of CTX-M-type ESBLs in China and India, it could be speculated that CTX-M-

type ESBLs are now actually the most frequent ESBL type worldwide.

The relationship between antibiotic consumption and occurrence of CTX-M-type

beta lactamases has not been studied, although the prevalence of the enzymes in

agents of community acquired that oxyiminocephalosporins available outside the

hospital (such as ceftriaxone) may be important. Interestingly, identical beta

lactamases have been discovered in widely separated parts of the world (for

example, CTX-M-3 has been discovered in Poland and Tai- wan), suggesting

independent evolution of these enzymes (Gniadkowski et. al., 1998; Yan et. al., 2000).

2.17.5. Inhibitor-Resistant Beta-Lactamases.

Although the inhibitor-resistant β-lactamases are not ESBLs, they are often discussed

with ESBLs because they are also derivatives of the classical TEM- or SHV-type

enzymes. In the early 1990s, β-Iactamases that were resistant to inhibition by

clavulanic acid were discovered. Nucleotide sequencing revealed that these enzymes

were variants of the TEM-1 or TEM-2 β-lactamase. These enzymes were at first given

the designation IRT for inhibitor-resistant TEM β-lactamase; however, they have been

subsequently renamed with numerical TEM designations. Inhibitor-resistant TEM β-

lactamases have been found mainly in clinical isolates of E, coli, but also in some strains

of K. pneumoniae, Klebsielta oxytoca, P. mirabihs, and Citrobacter jreundli. Although the

inhibitor-resistant TEM variants are resistant to inhibition by clavulanic acid and

sulbactam, thereby showing clinical resistance to the β-lactam, β-lactamase inhibitor

combinations of amoxicillin-clavulanate, ticarcillin-clavulanate, and ampicillin-

sulbactam, they remain susceptible to inhibition by tazobactam and subsequently the

combination of piperacillin and tazobactam . These β-lactamases have primarily been

detected in France and a few other locations within Europe in a recent survey of

amoxicillin-clavulanate-resistant E. coli in a hospital in France.

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2.17.6. Epidemiology of ESBLs in DFU.

Currently there is paucity of data on the epidemiology of ESBL producing organisms

isolated from DFU patients in the World and also from India. The extensive studies on

infection with ESBL producing organisms in DFU patients in India are scarce. Mathur et

al., from Chandigarh, India [Mathur et. al., 2002] have reported 68.5% of their DFU

isolates were ESBL producers. Babypadmini and Appalaraju (2004)have shown 40% of

K. pneumoniae isolates and 41% of E. coli isolates were ESBL producers in their study

from Vellore, India. The prevalence of ESBL was 6% in E. coli isolated from DFU patients

from Brazil (Armstrong and Lipsky, 2004). Gadepalli et al (2006) have reported that

54.5% E. coli isolates were ESBL producers in diabetic foot infections in Delhi, India. In a

recent study carried out at Shobha et al [2009] have reported 27.3% Klebsiella

pneumonia, 25.2% Escherichia coli, 21.42% Pseudomonas spp, 25% Enterobacter spp

and 17% Acinetobacter spp were ESBL producer.

There is paucity of data on molecular studies regarding the occurrence of ESBL bla

genes (CTX-M, TEM, SHV) from this country in relation to diabetic foot ulcer infection. In

India, the first report on the molecular detection of ESBL from clinical isolates was from

New Delhi, India the study on CTX-M-15 (Karim et al. 2001). bla SHV and blaTEM

producing K. pneumoniae was reported from two separate studies from Delhi on clinical

isolates(Grover et al. 2006; Lai et al. 2007). CTX-M15 was isolated from

Enterobacteriaceae family from three different medical centres from India (Ensor et al.

2006). Recently, Shahid M et al., (2011) detected blaCTX-M, blaTEM and blaSHV in

Enterobacteriaceae from North Indian clinical isolates. Presence of any bla genes (CTX-

M, TEM & SHV) was found in a total of 38.4% from 73 isolates. Many isolates showed

occurrence of genes in various combinations. CTX-M, TEM and SHV were noticed in

28.8%, 10.9% and 13.7% respectively.

2.17.7. Importance of ESBL detection in DFU isolates.

The empiric management of polymicrobial infections in DFU involves the use of a

combination of antibiotics that are effective against specific bacterial species, for

example, a combination of aminoglycosides and anti-anaerobic agents, such as

metronidazole or clindamycin [Ho et al., 1987]. However, to overcome the

incompatibility of certain antimicrobial agents and the resultant toxicity, separate

administration is sometimes necessary. The avoidance of potentially nephrotoxic agents

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is particularly important in diabetic patients because of the likely presence of renal

impairment. In addition to this, monitoring of pharmacokinetics is essential in order to

ensure that adequate therapy is given. Cost of drugs (acquisition, preparation, and

administration) also tends to be higher if combination regimens are given. A more

practical approach is to use a single parenterally administered antimicbial agent due to

the seriousness of such infections [DiPiro et. al., 1994]. The second-generation

cephalosporins also possess anti-anaerobic activity (notably cefoxitin and cefotetan)

provide one therapeutic option [Macgregor et al., 1992]. However, both these agents

can only be administered parenterally; if subsequent oral treatment is required, an

alternate agent must be prescribed. Another disadvantage of the second-generation

cephalosporins is that they are relatively poor in vitro activity against enterococci

[Karchmer et al., 1995]. The use of a beta lactam: beta-lactamase inhibitor combination

is another option. These agents possess a broad spectrum activity and provide coverage

against all potential pathogens, including anaerobes. The β-lactam: β-lactamase

inhibitor combinations ticarcillin–clavulanic acid and piperacillin–tazobactam

antibiotics have similar activity to that of sulbactam–ampicillin, which were

administered parenterally. The treatment requires that adequate tissue levels of

antibiotic are achieved to ensure eradication of the likely pathogens. Pharmacokinetic

studies have shown that concentrations of sulbactam–ampicillin in colonic tissue exceed

the MIC90 of Bacteroides fragilis [Martin et al., 1998], and levels in peritoneal fluid are

high enough to inhibit most susceptible β-lactamase-producing pathogens encountered

in intra-abdominal sepsis [Wise et al., 1983]. Similarly, sulbactam–ampicillin

penetration of the myometrium is sufficient to provide effective therapy for pelvic

infections [Schwiersch et al., 1986]. Penetration of infected tissue is particularly crucial

in diabetic patients because of peripheral vascular insufficiency. Seabrook et al. (1991)

reported that, after a single therapeutic dose of sulbactam–ampicillin, 42.8% of patients

studied showed level of drug higher than 10 mg/day in diseased soft tissue and this

combination is significantly superior to those achieved with either gentamicin or

clindamycin. The microbiological and pharmacokinetic properties of sulbactam–

ampicillin suggest its suitability for the treatment of mixed infections. Extensive clinical

evaluation has been conducted to confirm both the clinical and bacteriological efficacy

of sulbactam–ampicillin, and to demonstrate the cost-effectiveness of the combination.

The findings of these studies are summarized below. Infections in the feet of patients

Review of Literature

78

with diabetes mellitus are responsible for 20% of admissions in this patient group

[Sapico et al., 1989] and are a major cause of limb amputation [Grayson et al., 1994].

Sulbactam–ampicillin is an appropriate choice of treatment because of its efficacy and

relative lack of renal toxicity.

Four randomized trials were done to assess the efficacy of drugs in diabetic patients with infected foot ulcer. Two of these trials addressed non-limb threatening infections, third one addressed non-limb threatening but treatment resistant infection and the fourth one on invasive infection.

a) FIRST TRIAL: The first randomized trial to access the efficacy of beta lactam drugs

in the treatment of DFUs was done in 1990 by Lipsky et al. He randomized 56 patients

with infected lesions regardless of type or duration to oral Clindamycin or oral

cephalexin in an outpatient setting. After 2 weeks, no statistical significant difference

were found between treatment, either for response to infection or wound healing, the

latter occurring in 40 % of patients receiving clindamycin and 33% receiving

cephalexin.

b) SECOND TRIAL: Grayson et al., in the year 1994 randomized 93 patients to

intravenous imipenem/cilastatin or ampicillin/sulbactam. Patients had severe

infections of the lower extremities, threatening to the lower limb and identified by the

presence of cellulites, with or without ulceration or purulent discharge. Osteomyelitis

was diagnosed in 59(63%) patients. After 5 days, cure had been affected in 60% of the

ampicillin/ sulbactam group and 58% in imipenem/cilastatin group.

c) THIRD TRIAL: Chatelau et al., [1996] randomized 44 patients to oral amoxicillin

plus clavulanic acid or matched placebo. Patients had neuropathic ulcer of severity 1A

(superficial with/without cellulitis) to 2A (deeper, reaching to joints and tendons) on

Wagner and Harkless classification. At 20 days follow-up, no significant difference were

apparent between treatment and placebo in the numbers of ulcer healed or the mean

reduction in ulcer.

d) FOURTH TRIAL: Lipsky et al., [1997] randomized 88 patients to intravenous

Ofloxacin followed by oral Ofloxacin or intravenous ampicillin sulbactum followed by

oral amoxicillin clavulanate in patients who were hospitalized for soft tissue infections

that had not responded to outpatient management but which were not limb

threatening. At 28 days there were no statistically significant differences in the efficacy

of the two therapies. Cure occurred in 49% of the Ofloxacin group and 56% of the

amino-penicillin group.