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Review of Literature
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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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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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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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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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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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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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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20
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.
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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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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
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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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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
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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
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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)
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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.
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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
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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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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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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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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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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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54
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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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.