INTRODUCTION:

Diabetes mellitus is a severe and composite disease affecting almost all the crucial organs in the body [1].The metabolic deregulation associated with diabetes mellitus causes secondary pathophysiological changes in several organ systems that require a great burden on the individual with diabetes and on the health care system [2]. In recent years studies have accomplished the relationship of cardiovascular diseases type 2 DM and long term weight gain [3]. All forms of diabetes both genetic and acquire are characterize by hyperglycemia an absolute lack of insulin and the improvement of diabetes particular micro-vascular pathology in the retina, renal capillaries and peripheral nerves. Diabetes is

also associated with inappropriate and accelerated atherosclerotic macro-vascular disease affecting arteries that supply the heart, brain, and lower extremity [4]. Diabetes Foot Ulcers are complex chronic wounds which have a major long-term impact on the morbidity, mortality, resource exploitation and quality of patients [5].Treatment of these foot ulcers is challenging because of their multifactorial etiology and it places a high burden on patient’s healthcare systems and society [6].

Types of Diabetes

There are two main types of diabetes:

Type 1

Type 1 diabetes, insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes. These are mainly occurring in children’s and young adults, even though it can occur at some age. Type 1 diabetes may used for 5-10% of all diabetes in the United States. There does emerge to be a genetic component to Type 1 diabetes but the causes have so far so to be identified.

Type 2

Type 2 diabetes Non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes. [7] It may use for 90-95% of all diabetes. Type 2 diabetes mainly affects adults; on the other hand Type 2 has begun developing in children. There is a strong relationship between Type 2 diabetes, physical inactivity and obesity. [8]

Diabetes Mellitus:

The term diabetes is the full name diabetes mellitus. Diabetes mellitus is derived from the Greek word diabetes meaning siphon to pass through and mellitus the Latin word mellitus meaning honey or sweet. Diabetes mellitus is a chronic lifelong condition that affects the body’s ability to use the energy found in food. There are three major type of diabetes type 1 diabetes, type 2 diabetes and gestational diabetes. All types of diabetes mellitus have something in common. Usually body breaks down the sugars and carbohydrates eat into a special sugar called glucose. Glucose fuels the cells in the body but the cells is need insulin a hormone in bloodstream in order to take in the glucose and use it for energy with diabetes mellitus.[9]

Pathophysiology of Diabetes Foot Ulcers:

Diabetes foot ulcer is characterized in a typical triad of neuropathy, ischemia, and infection [10] due to the impaired metabolic mechanisms in Diabetes mellitus, there is an improved risk of infection and poor wound healing due to a series of mechanisms which include decreased cell and development cause response, diminished peripheral blood flow and decreased local angiogenesis [11]. So, the feet are given to peripheral vascular disease, damage of peripheral nerves, irregularity ulcerations and gangrene. These changes occur at a biomolecular level and are caused by hyperglycemia, which leads to the development of neuropathy pathogenetic role of a number of mechanisms that lead diabetic persons to develop wounds. The major mechanisms are nerve hypoxia/ischemia, Auto oxidative stress, Polyol pathway over activity, Increased advanced glycation end-products, Deficiency of gamma linolenic acid, Protein Kinase C, especially B-isoform increase, Cytokines dysfunction, disorders of collagen molecules endothelial dysfunction, Mitochondrial dysfunction, Growth factors deficiency, Alteration of the immune mechanism. [12]

Diabetes Foot Infection:

Diabetic Foot Infections (DFIs) are defined as a clinical syndrome characterized by local findings of inflammation occurring in a site below the malleoli in a person with diabetes estimate of the incidence. Diabetic foot infections range from a lifetime risk of 4% in all persons with diabetes to 7% yearly in patients treated in a diabetic foot centrally [13]. In diabetic patients with foot wound the physician should perform a systematic examination to determine if the wound is infected and the degree of severity of understood infection. The examination should start with critical signs, consciousness, complete review of the limb to the foot end, always removing the patient’s shoes and socks, and should include a neurological, vascular, dermatological and muscle skeletal examination to determine the external characteristics of the skin the shape and structure of the foot and alterations in the fingers, protruding metatarsal, heel, etc.

Figure-1:- Infections in diabetic foot ulcers.

The most diabetic foot infections occur in a neuropathic or neuroischaemic ulcer, which serves as a point of entry for pathogens with the exception of erysipelas and posttraumatic infection [14] diabetic foot infections are almost always epiphenomena i.e. the consequence of progressive peripheral polyneuropathy with associated loss of protective sensation coupled with gait disorders anterior displacement of weight-bearing during walking [15] with reduced mobility, and arterial insufficiency in a mostly mature patient population [16]. Vascular disease, mostly in the form of occlusive atherosclerotic disease of the arteries below the knee, sometimes accompanied by small vessel dysfunction [17], It can cause ischemic ulcers and may contribute to elevated plantar pressures and to prolonged duration of foot-to-floor contact [18].

Location of Foot Ulcer in Diabetes:

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 [19, 20]. Ulcer severity is more important than site in determining the final outcome [21, 22]. The details of site of ulcer and its treatment. Several reported that several factors were contributing to the outcome of Diabetes foot ulcer which is irrespective of similar type of care treatment [23-27] 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 [28]. Patients in all these studies were receiving the similar ulcer care, including off-loading, wound debridement and healing treatment [29-32].

Risk Factors for Diabetic Foot Ulcer:

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 both baseline vibration perception threshold and combined score of reflexes and muscle strength are significant predictors of ulcer [33] A significant correlation between absence of switch pulse or history of peripheral arterial revascularization with foot ulcer. [34, 35]. Only a single study reported that foot deformity as a significant risk factor for diabetic foot ulcer. [36]

Diagnosis and Physical Examination:

The foot examination should begin with noting the condition of the skin involved in and surrounding the ulcer. The key features of the ulcer to note during exam are Depth, Location, Drainage, and Sign of infection Presence of viable and nonviable gangrenous tissue. Osteomyelitis is present in approximately 70% of ulcers that can be probed to bone. [37] A comprehensive neurovascular examination should be performed with careful attention to pulses and level of neuropathy, which can be determined with a standardized ten gauge monofilament.

Examination of ulcer:

A sterile stainless steel probe is used for assessing the ulcer to determine the depth and if there is sinus tracts present [38]. The location, size, shape, depth, base and margins of the ulcer should be examined clinically. Presence of granulation tissue or slough should be looked for in the floor of the ulcer to determine subsequent management diagnosing a soft tissue infection in patient with diabetes is sometimes difficult as the signs of inflammation of the overlying ulcer may be absent. The infection is mainly diagnosed based on presence of clinical signs and symptoms such as redness, warmth, tenderness, purulent secretions and fever [39].Palpation of the bone at the base of the ulcer with a sterile, blunt stainless steel probe has been suggested as positive predictor of underlying Osteomyelitis [38].

Neurological testing

Sensory neuropathy can be tested by using monofilaments and biothesiometer. Semmes Weinstein monofilaments are reported to be easy to use and help in predicting the risk of ulceration and amputation [40, 41] suggested annual testing of all patients with diabetes with a nylon monofilament to detect peripheral neuropathy. Pain sensation should be tested as well. The Heart Rate Variability (HRV) with deep breathing or orthostatic blood pressure is measured to detect autonomic neuropathy [42] and any decrease or absence of HRV is considered the earliest sign of autonomic neuropathy in DM [43]. Specialized tests for sudomotor dysfunction include thermoregulatory sweat testing, quantitative sudomotor axon reflex testing, silicone impressions, the Sympathetic Skin Response and the quantitative direct and indirect axon reflex testing. These tests can be used in various combinations to localize the lesion of autonomic dysfunction (pre-ganglionic or post-ganglionic) [44].

Laboratory investigations

The standard procedure involves measuring blood glucose level and urine for glucose and ketones. Other investigations like full blood count, blood urea, electrolytes, and creatinine levels should be monitored regularly. Glycosylated hemoglobin is important to gauge the patient’s overall glycemic control the mean blood sugar concentration best over previous weeks to months [45]. Hepatic and renal function tests are necessary for monitoring the patient’s metabolic status. It can be done to assess the presence and response to treatment of infections like osteomyelitis [46]. Routine wound cultures are not recommended since all wounds harbor microorganisms [47]. However in the presence of invasive infection, cultures from the deeper tissue will help to identify the causative microorganisms.

Treatment of Diabetes foot ulcers:

Pharmacological therapy

Individual patient education improved diabetes knowledge and self-management activities have improved medication adherence to oral diabetic medications in case-controlled trial.[48] Diabetic neuropathies resulting from chronic sensory motor distal symmetrical polyneuropathy poses challenges for neuropathic pain. Significant atherosclerotic risk factors should be addressed to reduce risk of development of concurrent peripheral vascular disease or critical limb ischaemic. Main key strategies include smoking cessation and use of pharmacological aids such as nicotine replacement and statins irrespective of cholesterol levels and antiplatelet medications. Duration of treatment ranged from two weeks up to two months depending on the severity of the underlying infection.

Improving vascularisation

Revascularization of critically ischemic legs results in increased perfusion after the procedure which in turn is associated with a further reduced amputation rate.

Debridement

Repeated sheer pressure on the diabetic foot results in callus formation. The removal of necrotic and hyperkeratotic tissue either in the form of superficial ulcer Debridement or selective sharp debridement thus promotes better wound healing. Deep wounds, specifically those with bone and soft tissue involvement, require more aggressive debridement with some involving surgery.[49-50]

Wound dressings

Dressings offer an external protection and barrier to external forces and contaminants while promoting absorption of exudates around the ulcer site. There are a variety of dressing types available along with increasingly advanced methods of promoting wound healing [51] Despite further advancements in wound dressing there has however been limited evidence to suggest that moist dressings are more effective than ‘dry’ dressings or vice versa.[52] Silver impregnated dressings have not been shown to be more effective in treating diabetic foot ulcers in randomized controlled trials than dressings for treating any other wound.[53]

Negative pressure wound therapy:

Targeted negative pressure wound therapy is another increasingly common method used in the management of diabetic foot ulcers primarily involving the removal of wound fluid through a sealed vacuum. This is aimed at improving tissue perfusion and in the promotion of formation of granulation tissue, and often a shorter treatment in comparison with ulcers treated with traditional gauze dressing. However, a Canadian evidence-based study revealed no statistically significant difference between negative pressure wound therapy and the standard wound care in length of time to complete wound closure.[54]

Growth Factors:

In the broadest sense, the term growth factors is used for substances with chemotactic properties to attract pro-inflammatory cells and fibroblasts into the wound, to promote cellular proliferation, angiogenesis, production and degradation of the extracellular matrix, and synthesis of cytokines and growth factors by neighboring cells. However, these multiple effects are not stimulant for all cells. For instance, TGF-beta stimulates the mitosis of cells deriving from mesoderm (fibroblasts), while inhibits the mitosis of cells derived from ectoderm (keratinocytes). The most important growth factors for the wound healing are families of PDGF, EGF, FGF, TGF and IGF [55, 56]. The binding of growth factors to cell surface receptors initiates the specific cell transduction pathways. This process produces changes in gene expression what leads to new protein synthesis, changes in cellular activity or proliferation. Growth factors promote, generally, wound healing via several mechanisms: chemotaxic activities attraction of inflammatory cells and fibroblasts into the wound, stimulation of cellular proliferation, angiogenesis (in growth of new blood vessels), production and degradation of extracellular matrix, and synthesis of cytokines and growth factors by neighboring cells.

Wound Healing:

Wound healing is a complex biologic process that optimally leads to restoration of tissue integrity and function. The process is very orderly and well coordinated. Generally, the healing process progresses through three characteristic stages: inflammatory phase, proliferative (granulation tissue formation), and repair (scar formation stage) and remodeling stage. Inflammation begins immediately upon tissue injury, thereafter migration of fibroblasts and other cells into the site of injury, as well as the initial scar formation initiate the proliferative stage. Finally after initial scar formation, proliferation and neovascularization cease, the wound enters the terminal remodeling stage. The stages of wound healing overlap temporarily and the entire process can last for several months. Usually, the inflammation process lasts several days, the proliferative stage several weeks, whereas the remodeling phase several months.

Acute Wounds and Growth Factors:

Acute wounds are generally less complex than chronic wounds. The spontaneous healing of acute wounds does not represent a serious problem in clinical practice. However, the rate of spontaneous healing is not the highest rate that can be achieved. For instance, topical application of EGF, TGF-alpha, PDGF and IGF accelerates the rate of epidermal full or partial thickness wound healing in experimental animals approximately for 20% to 30% than vehicle controls, whereas topical EGF accelerates the epithelial wound healing in humans by average of 1.5 day [57]. It appears, therefore, that the amounts of peptide growth factors are probably not optimal in spontaneous healing skin wounds. The clinical benefit, therefore, of topical growth factors in healing wounds would be negligible in a small wound, but not in large size wounds. Interestingly, apart from qualitative differences in growth factors, quantitative changes also exist in fluids of acute and chronic healing wounds. As a rule, contrary to chronic healing wounds, inflammatory cytokines, proteases and growth factors occur in relatively small amounts in spontaneously healing wounds.

Chronic Wounds and Growth Factors:

When the normal healing process is interrupt a chronic wound occurs. Chronic skin and soft tissue wounds are classified as diabetic foot ulcer, decubitus ulcer, venous stasis and ischemic ulcer caused by arterial insufficiency. Many different causes underlay the chronic wounds. The most common causes are similar in that each is characterized by one or more persistent stimuli, repeated trauma, ischemia or low grade bacterial contamination [58-61]. In contrast to acute wounds, in chronic wounds the levels of inflammatory cytokines are very high, whereas the amounts of growth factors are decreasing. Multiple possible causes, such as the presence of bacteria and their end toxins, degradation products of platelets and degradation products of extracellular matrix trigger the production of inflammatory cytokines like TNF and interleukins (IL-1, IL-6)[55,56]. The increased production of these inflammatory cytokines leads to a concomitant elevation of production of proteases. The increase of proteases, including matrix metalloproteinase’s, leads to degradation of growth factors. When the growth factors are drastically reduced the communication between the various cells participating in the process of healing stops and the wound healing is delayed. Therefore, it is not surprising that non healing wounds have been described as being „stacked “in the inflammatory stage. In fact, healing process only after the inflammation is controlled [62-64].

Becaplermin Chemistry (Recombinant Platelet Derived Growth Factor):

Platelet Derived Growth Factor (PDGF) is a dimer consisting of A and/or B chains held together by a disulfide bond. From human platelets AA, BB and AB isomers have been isolated. The most potent isomer is BB [55, 65, 66]. Using recombinant DNA technology a homodimeric protein was produced by inserting the gene for the B chain of human PDGF into the yeast Saccharomyces cerevisiae. The recombinant PDGF-BB (rh-PDGF-BB) of becaplermin has biological activity similar to endogenous PDGF-BB [55, 67].

Synthesis, release and mechanism of action via receptors:

Two cell surface PDGF receptors have been identified to date: alpha- and beta-PDGF receptors. The alpha- PDGF receptor is non-specific and binds to all isoforms of PDGF. On the other hand, beta-PDGF receptor selectively binds only to PDGF-BB isoform. The most common type of receptors is beta PDGF cell surface receptor [67, 68].

Synthesis and release

Platelets, macrophages, endothelial cell, fibroblasts, keratinocytes, smooth and skeletal muscle cells and astrocytes synthetize and release PDGF [64, 66].

Mechanism of action

Becaplermin (Regranex Gel) has biological activity similar to that of endogenous platelet-derived growth factor, which includes promoting the chemotactic recruitment and proliferation of cells involved in wound repair and enhancing the formation of granulation tissue. After binding of PDGF to its receptor dimerization leads to tyrosine auto-phosphorylation, intracellular transduction, induction of early response genes (c-myc, c-fos) and the range of cellular responses, such as new protein synthesis, changes in cellular activity and proliferation [57,63, 69].

Figure 2: Mechanism of action of Becaplermin.

Biological activities of Becaplermin:

Biological activities of becaplermin are similar to that of endogenous PDGF-BB promoting the chemotaxic recruitment and proliferation of cells sub serving the wound-healing cascade [65,76, 67]. The basic effect of PDGF-BB and becaplermin is the promotion of wound healing. PDGF-BB is actively involved in all stages of wound healing process. The promotion of wound healing process is ascribed [55, 69] to chemotaxic effect (migration of neutrophils, monocytes, fibroblasts and smooth muscle cells into wound), stimulation the synthesis of fibroblasts and extracellular matrix, and stimulation the proliferation of smooth muscles. Other (non-dermal) effects include: anti-inflammatory effect, smooth muscle hypertrophy of uterus, lens growth and transparency and central nervous system gliogenesis.[69]

Pharmacodynamics and Pharmacokinetics:

Clinical pharmacodynamic studies have not been conducted.

Ten patients with Stage III or IV (as defined in the International Association of Enterostomal Therapy (IAET) guide to chronic wound staging,1,2 lower extremity diabetic ulcers received topical applications of becaplermin gel 0.01% at a dose range of 0.32–2.95 µg/kg (7µg/cm2) daily for 14 days. Six patients had non-quantifiable PDGF levels at baseline and throughout the study, two patients had PDGF levels at baseline which did not increase substantially, and two patients had PDGF levels that increased sporadically above their baseline values during the 14 day study period.

Nonclinical toxicology:

Carcinogenesis, mutagenesis, impairment of fertility

Becaplermin was not genotoxic in a battery of in vitro assays (including those for bacterial and mammalian cell point mutation, chromosomal aberration, and DNA damage/repair). Becaplermin was also not mutagenic in an in vivo assay for the induction of micronuclei in mouse bone marrow cells. Carcinogenesis and reproductive toxicity studies have not been conducted with REGRANEX Gel.

Side effect

Common side effects of REGRANEX include:

Red skin rash, burning at the application site

Indications and usage

REGRANEX Gel contains becaplermin, a human platelet-derived growth factor that is indicated for the treatment of lower extremity diabetic neuropathic ulcers that extend into the subcutaneous tissue or beyond and have an adequate blood supply. Regranex Gel is indicated as an adjunct to, and not a substitute for, good ulcer care practices.

Limitations of use

The efficacy of REGRANEX Gel has not been established for the treatment of pressure ulcers and venous stasis ulcers. The effects of REGRANEX Gel on exposed joints, tendons, ligaments, and bone have not been established in humans. REGRANEX Gel is a non-sterile, low bioburden preserved product. Therefore, it should not be used in wounds that close by primary intention.

Dosage and administration

For topical use; not for oral, ophthalmic or intravaginal use.

Contraindications

Known neoplasm(s) at the site(s) of application

Warnings and precautions

Malignancies distant from the site of application have been reported in both a clinical study and in postmarketing use. REGRANEX Gel should be used with caution in patients with a known malignancy.

Adverse reactions

Erythematous rashes occurred in 2% of patients treated with REGRANEX Gel

Drug interactions

It is not known if REGRANEX Gel interacts with other topical medications applied to the ulcer site. The use of REGRANEX Gel with other topical drugs has not been studied.

Use In Specific Populations:

Pregnancy

Pregnancy Category C. There are no adequate and well-controlled studies in pregnant women treated with REGRANEX Gel. REGRANEX Gel should be used during pregnancy only if the potential benefit justifies the potential risk to the fetus. Animal reproduction studies have not been conducted with REGRANEX Gel.

Nursing mothers

It is not known whether becaplermin is excreted in human milk. Because many drugs are secreted in human milk, caution should be exercised when REGRANEX Gel is administered to nursing women.

Pediatric use

Safety and effectiveness of REGRANEX Gel in pediatric patients below the age of 16 years have not been established.

Geriatric use

Among patients receiving any dose of REGRANEX Gel in clinical studies of diabetic lower extremity ulcers, 150 patients were 65 years of age and older. No overall differences in safety or effectiveness were observed between patients < 65 years of age and patients ≥ 65 years of age. The number of patients aged 75 and older were insufficient (n=34) to determine whether they respond differently from younger patients.

Over dosage

There are no data on the effects of becaplermin overdose.[70]

Clinical use:

Becaplermin is applied topically as a gel (0.01%; 100 μg/g) once a daily to a clean, dried wound in a thin layer for 20 weeks or complete healing. The patient at home can apply the gel. A moist healing environment, debridement of all necrotic debris and callus, control of infection and pressure relief of affected area are essential conditions for the application of becaplermin gel. The thin layer of becaplermin gel has to be covered with a moist gauze dressing. Debridement, a sharp incision with subsequent bleeding, at the site of the wound is a fundamental condition since there is evidence that debridement removes cells no longer responding to the action of growth factors, as well as that platelets from the bleeding surface release PDGF into the wound. In conclusion, becaplermin (rh-PDGF-BB) is the first commercially available growth factor, a new highly effective and safe adjuvant, topical drug treatment of chronic neuropathic diabetic foot ulcer if used under optimal wound-healing (debridement of all necrotic debris and callus, control of infection and pressure relief of affected area) environment. Serious unwanted effects have not yet been reported from the topical application of becaplermin. [71].

Factors affecting healing of diabetic foot ulcers:

Factors affecting healing of diabetic foot ulcers that included the location of ulcer, duration of diabetes, ulcer duration, the presence of heart failure and peripheral arterial disease. The proximal location of the ulcer corresponded with maximal healing time with no difference in healing times between plantar and non plantar ulcers. Sheehan et al. [72] noted that the percentage change in foot ulcer area after 4 weeks can predict of healing at the end of 12 weeks and can be used as an early indicator of unresponsiveness to treatment. Increased size and depth of ulcer have been associated with poor healing [73].

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 [74, 75] 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 [76, 77].

Antibiotic Therapy:

The antibiotic therapy is usually 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 three to five days. Most of the patients are shifted to oral antibiotics [78]. Oral antibiotic therapy is less expensive and more convenient. For mildly infected patients, tropical therapy is the better option of treatment. This treatment has several advantages, including high local drug levels avoidance of system in adverse effect [79]. The patients with peripheral vascular disease, therapeutic antibiotic concentrations with many agents are often not achieved in tissue even while the serum concentrations are adequate [80]. In one procedure called retrograde venous perfusion, antibiotic solution is 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 [81].

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 and 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.

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 [82] and long term outcome are reported to be achieved in 80 % of patients. In many patients, above ankle amputations are avoided and the uses of aggressive antibiotic therapy with minor and major surgical intervention are required [83].

CONCLUSION:

Optimal treatment of diabetic foot infections requires pathogen specific antibiotic therapy and proper identification is indispensable. Culture-based techniques select for pathogens that flourish under the typical nutritional and physiological enviroment of the diagnostic microbiology laboratory, not necessarily the most abundant or clinically important organism. In the past decade the studies done by molecular techniques have raised the doubts on wound culture results. Sequence analysis of hypervariable portions of 16S rRNA region have vastly revealed more complex bacterial communities than identified by conventional methods. So it could be expected that in near future more innovation in molecular microbiological field will overcome all the low loops of culturing techniques and will hold a promise in proper management of DFUs.

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Received on 21.07.2017 Modified on 01.09.2017

Res. J. Pharmacology & Pharmacodynamics.2017; 9(3): 157-166.

DOI: 10.5958/2321-5836.2017.00027.1