Medical Journal of Dr. D.Y. Patil Vidyapeeth

: 2020  |  Volume : 13  |  Issue : 5  |  Page : 447--453

Prevalence of microbial agents associated with chronic nonhealing ulcers: A cross-sectional study

Pritilata Panda, Sarvodaya Tripathy 
 Department of Microbiology, P.R.M. Medical College and Hospital, Baripada, Department of Microbiology, M.K.C.G. Medical College, Berhampur, Odisha, India

Correspondence Address:
Sarvodaya Tripathy
Department of Microbiology, M.K.C.G. Medical College, Berhampur, Ganjam, Odisha


Background: Chronic nonhealing ulcers (NHUs) are responsible for considerable morbidity worldwide. Polymicrobial flora has been associated with most of the chronic ulcers. Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacteriaceae, Anaerobes, Mycobacteria, and Fungi have been shown to infect chronic NHUs. Objectives: The objective of the study was to evaluate the microbial agents associated with chronic NHUs. Materials and Methods: The study was a cross-sectional observational study conducted over 2 years. Basing on the inclusion criteria sample were collected from 168 patients. The standard protocol for the identification of microorganisms was followed. Results: Of the 168 cases examined 128 (76.2%) were male and 40 (23.8%) were female. Most of the cases were 41–60 years old. One hundred and thirty-two microbial isolates were obtained from 77 patients. In 32 patients' infections were monomicrobial and in the rest 45 infections were polymicrobial. A total of 112 out of 132 isolates obtained in the study were bacteria (both aerobic and anaerobic). Mycobacterial isolates were 11 and fungal isolates were 9 in number. Conclusion: The prevalence of microbial infections associated with a chronic NHU in this study population was 45.8%. The most common group of microbial agents were aerobic bacteria, S. aureus being the most common agent.

How to cite this article:
Panda P, Tripathy S. Prevalence of microbial agents associated with chronic nonhealing ulcers: A cross-sectional study.Med J DY Patil Vidyapeeth 2020;13:447-453

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Panda P, Tripathy S. Prevalence of microbial agents associated with chronic nonhealing ulcers: A cross-sectional study. Med J DY Patil Vidyapeeth [serial online] 2020 [cited 2020 Dec 2 ];13:447-453
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Full Text


Chronic ulcers are defined as “wounds which have failed to proceed through the orderly process that produces satisfactory anatomic and functional integrity.”[1],[2] An ulcer becomes chronic, when the normal process of healing is hampered due to various intrinsic (immune status, nutritional status, medical comorbidity, site, and nature of ulcers) and extrinsic factors (infection and treatment offered).[3],[4] Many authors have considered 4–6 weeks as the time limit to define an ulcer to be chronic.[5]

Chronic nonhealing ulcers (NHUs) are responsible for considerable morbidity and a significant cost of health-care worldwide.[5] In the absence of proper wound management, an infection can lead to serious complications such as amputation of limb, septicemia, and even death.[6],[7] In developing countries, approximately 1%–2% of the population is expected to experience a chronic wound during their lifetime.[5],[8] NHUs commonly occur in the lower extremities. Vascular insufficiency and neuropathy are the most common causes of ulcers in this part of the body.[3] In an Indian study, Gupta et al. also found 74.2% of the chronic ulcers to occur in lower extremities.[9]

Studies report that polymicrobial flora (a mixture of Gram-positive and Gram-negative as well as aerobes and anaerobes) has been associated with most of the chronic ulcers.[10],[11],[12],[13] The most common bacterial cause of wound infection leading to its chronicity is Staphylococcus aureus as found in various studies.[12],[14],[15],[16] Other common bacteria that have been implicated in the chronicity of ulcers include Pseudomonas spp. and the Enterobacteriaceae family. Wound infecting bacteria such as Staphylococcus, Streptococcus, and Pseudomonas can produce exotoxins that lead to extensive destruction of host tissue.[2] These also disrupt normal cellular metabolism causing further tissue necrosis. Gjødsbøl et al., Bowler and Davies, Johnson et al., Sapico et al., etc., highlight the presence of anaerobic bacteria in chronic NHUs.[12],[14],[17],[18] Nearly every pathogenic species of nontuberculous mycobacteria can cause skin and soft-tissue infections, but rapidly growing mycobacteria (Mycobacterium fortuitum, Mycobacterium chelonae, and Mycobacterium abscessus), Mycobacterium marinum and Mycobacterium ulcerans are the most commonly involved.[19] Fungi also are seen to be associated with chronic NHUs.[20] Patients with diabetes mellitus are at increased risk of developing foot ulcers and fungus may be an important isolate in these patients.[21] In diabetic foot ulcers, mixed infection of both bacteria and fungi have been reported. The percentage of ulcers with mycotic superinfection ranges from 1% to 24%.[22] Studies on the prevalence of microbial agents in various types of chronic ulcers in the Indian population are scarce. The Indian population is mostly dependent on farming and is exposed to significant climatic variations. There are major health issues such as malnourishment, substance abuse, accidents, infectious diseases, and diabetes, which are independent risk factors for chronic NHUs. Understanding the causes of chronic ulcers in the Indian population and microbial agents associated with them will give us an insight into this problem, which will sensitize the clinicians in providing appropriate care.

Researchers studied the association of various pathogens with chronic NHUs through prospective as well as cross-sectional studies. However, the paucity of time in time-bound studies often tempts researchers to follow a cross-sectional research design.

The management of chronic NHUs is a challenging and costly affair.[23] The presence the above mentioned risk factors, which are commonly seen in developing countries like India, makes the challenge more grievous and priority for intervention. Persistence of chronic NHUs may have several after effects, which can be physical, psychological, functional, and financial.[24] Hence, it is worth studying the causes of chronic NHUs and the microbial agents associated with them in our region, not only for the sake of limiting the burden of care and quality of life but also preventing its aftereffects. In our study, we attempted to see the prevalence of the various microbial agents associated with chronic NHUs in patients attending a tertiary care center of coastal Odisha.

 Materials and Methods

It was a cross-sectional, observational study in the clinical sample of patients with chronic NHUs in a tertiary care hospital of coastal Odisha. This study was conducted under naturalistic conditions. The study was conducted from September 2014 to August 2016 (2 years).

Inclusion criteria

Patients with chronic ulcers persisting for 4 weeks or more were included in the study.

Exclusion criteria

Critically ill and debilitated patients were excluded from the study.

The study group included both outpatients and inpatients from the departments of surgery, dermatology, general medicine, and orthopedics. A total of 168 patients were examined.

Demographic data, clinical and laboratory reports were collected and recorded in a standard format for each patient. The necessary ethical clearance was obtained from the institutional ethics committee.

Sample collection

Ulcers were irrigated thoroughly using normal saline and cotton. Samples were collected in the form of swabs and aspirates. Swabbing was done from the deepest part of the ulcer. Deep-seated wounds were aspirated using a sterile syringe. If the sample were collected in the indoor ward, they were transported immediately to the microbiology laboratory and processed. Robertson's Cooked Meat (RCM) broth was used as transport media from indoor patients.

Sample processing

One swab or a small amount of aspirated pus was used for the preparation of slides, for Gram stain, Ziehl–Neelsen stain, and potassium hydroxide (KOH) mount.

For aerobic bacterial isolation, 5% blood agar (BA) and MacConkey Agar (MCA) were inoculated. Inoculations were also made into Brain Heart Infusion (BHI) broth. After inoculation of the sample, BA was incubated at 37°C in candle jar, MCA plate was incubated aerobically at 37°C. In the absence of any growth subculturing on to BA and MCA was done from the BHI broth and observed for growth on the next day. Any growth was then processed for identification using enzymatic and biochemical tests. Only after 48 h, the results for aerobic bacterial growth were declared negative. Antibiotic susceptibility testing was done for aerobic bacteria using Muller–Hinton plates by Kirby–Bauer Disc diffusion method.

For anaerobic bacterial isolation, the sample was inoculated on nonselective and selective Brucella BA medium and preserved in RCM. The Brucella BA plates were subjected to anaerobic incubation using the GasPak system, at 37°C. Subculture from RCM was done after 48 h (of its inoculation) on to nonselective and selective Brucella BA plates. A metronidazole disc (5 μg) was placed in the junction of the first and second sets of streaking lines in all plates. Bacterial isolates were then identified using the standard biochemical tests.

For suspected fungal etiologies Sabouraud's Dextrose Agar tubes were inoculated in pairs and incubated at 25°C and 37°C. Yeasts were subjected to a germ tube test. Further identification was done using Candida, chrome agar, and cornmeal agar. For filamentous fungi, slide culture was done and lactophenol cotton blue mount was used for identification.

For suspected mycobacterial etiologies readymade Lowenstein–Jensen (LJ) medium was inoculated. The standard protocol for the identification of mycobacteria was followed. Rapid growers were further identified by growth on MCA, nitrate reduction, sodium chloride tolerance, and 3-day arylsulfatase test.

All the processing was done in Class II biosafety cabinet.

Statistical methods

The data were analyzed in terms of percentages and proportions. A descriptive analysis of the content was done.

Ethical issue

The study was approved by the Institutional Ethical Committee of MKCG Medical College, Brahmapur, Orissa (Certificate No 221/Chairman IEC).


Of the 168 cases examined 128 (76.2%) were male and 40 (23.8%) were female. Most of the patients were 41–60 years old. Patients from rural areas constituted 59.5% of the cases and the rest 40.5% belonged to urban areas. Out of all the cases 92 belonged to lower socioeconomic status (54.8%), while only 4.1% belonged to the upper socioeconomic status. Farmers or laborers amounted to 52 (30.9%) cases which were the most common occupation among the cases. A history of tobacco and alcohol use was observed in 59.5% (n = 100) and 38.1% (n = 64) cases, respectively. Comorbidities such as tuberculosis, diabetes, and hypertension were seen in 14.3% (n = 24), 35.1% (n = 59), and 23.8% (n = 40) of the cases, respectively. The mean duration of the ulcers in these patients was 2.924 ± 3.334 years. In 104 (61.9%) cases, the duration of the ulcer was in between 1 and 5 years. The maximum duration of the persistence of ulcers in our study was found to be 22 years. The most common location of the ulcers was lower extremities, which were affected in 114 (67.9%) cases. Single lesions were present in 148 (88%) cases while the rest 20 (12%) had more the one lesions. Discharge from the ulcer was present in 114 (67.9%) cases. Microbial isolates were obtained from 56% of the ulcers having discharge while from 9% of the ulcers without discharge. Of the rest 54 (32.1%) patients in whom frank discharge was not associated with ulcers, microbial agents were isolated in 5 (2.97%) and one of these was a mycobacterial isolate and thus cannot be overemphasized. Most of the patients included in the study were referred from the department of surgery 68 (40.5%). This was followed by the departments of dermatology, general medicine, and orthopedics in that order. Of the 168 patients included in the study, microbial isolates were obtained from 77 (45.8%). A total of 132 microbial isolates were obtained from these 77 patients. Of these 132 isolates, 66 (50%) isolates were from the patients referred from the department of surgery only.

Of the 77 patients in whom microbial isolates were obtained, in 41.56% (n = 32) cases, infections were monomicrobial and in 58.44% (n = 45) cases infections were polymicrobial. Monomicrobial infections caused by aerobic bacteria constituted of 81.52% of all monomicrobial infections (n = 26). Of the mixed isolates which were 100 in number, the most common combinations were that of two aerobic bacteria followed by an aerobe with an anaerobe.

The most common microbial isolates obtained in the study were bacteria (84.8%), amounting to a total of 112 of 132 isolates. Among all the bacterial isolates 83.93% were aerobic bacteria (n = 94) and 16.07% were anaerobic bacteria (n = 18). Of all isolates, mycobacteria were obtained in 8.33%, amounting to a total of 11 of 132 isolates and fungi were obtained in 6.81%, amounting to a total of 9 of 132 isolates.

As depicted in [Table 1] The bacterial isolates constitute 84.85% (60.61% aerobic bacteria and 24.24% anaerobic bacteria) of all microbial isolates from the clinically diagnosed cases of diabetic foot ulcer. The bacterial isolates constitute 94.44% (72.22% aerobic bacteria and 22.22% anaerobic bacteria) of all microbial isolates from the clinically diagnosed cases of vascular ulcers (varicose ulcers and ischemic ulcers). Traumatic ulcers followed by diabetic ulcers were the largest single group of ulcers found in our study. Of the 47 traumatic ulcer cases, 29 were due to road traffic accident or major fall whereas 18 were following surgical procedures (that had initially healed in postoperative period). Bacterial isolates constitute 80.49% (70.73% aerobic and 9.75% anaerobic) of all microbial isolates obtained from traumatic ulcer cases. Other causes included – chronic osteomyelitis (n = 12); chronic folliculitis (n = 12); chronic dermatitis (n = 7); madura foot (n = 6); actinomycosis (n = 2); ruptured cold abscess and cervical lymphadenitis (n = 10); sinus discharge (n = 4); and NHUs of unknown etiology (n = 9). Of all the microbial isolates obtained from these patients, 85% (80% aerobic and 5% anaerobic) were bacterial.{Table 1}

The most common microbial isolate was S. aureus accounting for 19.69% (n = 26) of all isolates. Among the aerobic bacterial isolates (n = 94), the most common (27.67%) bacteria obtained was S. aureus followed by Pseudomonas aeruginosa (20.21%). Enterobacteriaceae constituted 30.85% of all aerobic bacteria, Escherichia coli (10.64%) being the most common in the group. Other members of Enterobacteriaceae isolated were Klebsiella pneumoniae (9.57%), Proteus spp. (9.57%), and Citrobacter freundii (1.06%). Staphylococcus epidermidis (8.53%), Enterococcus faecalis (6.38%), Acinetobacter baumannii (4.25%), and β hemolytic Streptococci (2.12%) were other isolates. The most common anaerobic bacteria isolated were Peptostreptococcus spp. contributing to 7.4% followed by Bacteroides fragilis which was 6.3% of all bacterial isolates in the study. Other anaerobes obtained in our study were Clostridium sporogenes followed by Clostridium perfringens and Prevotella spp.

A positive acid-fast bacillus stain was seen in 16 cases, however, in five-slide positive cases, no growth was obtained on any of the LJ-culture media tubes after an incubation up to 8 weeks. Among the 11 isolates of mycobacteria eight were M. fortuitum (72.73%) and three were M. chelonae (27.27%).

Gram stain and KOH mount had shown fungal elements in 12 cases. Fungal growth was obtained in 9 (5.36%). Out of the nine fungal isolates obtained 88.88% (n = 7) were yeasts and 22.22% (n = 2) were filamentous fungi. Candida albicans (4 out of 7) was the most common isolate followed by Candida tropicalis (2 out of 7) and Candida parapsilosis (1 out of 7). The two filamentous fungi isolated were Fonsecaea pedrosoi and Aspergillus fumigatus.


Most of the patients in our study were male. The male: female ratio in our study was found to be 3.2:1. Various international studies have shown a relatively higher prevalence of chronic NHUs in females especially in the elderly population.[9],[25],[26] However, Indian studies have found a higher prevalence of chronic NHUs in males. Saraf et al., reported male: female ratio of 5.7:1, whereas Gupta et al., reported the ratio to be 2:1.[9],[27] A hospital-based Chinese study also found male predominance in cases of chronic ulcers.[28]

The average age of the patients in our study was 42.39 ± 18.94 years. The minimum age of patient recorded was 9 years and the maximum age was 92 years. A total of 64 (38.1%) patients belonged to the age group of 40–60 years followed by 52 (30.95%) in the age group of 21–40 years. Several international studies have shown the elderly age group to be the most common group presenting with chronic NHUs.[3],[29],[30] In our study, however, we found 140 patients (83.33%) under the age of 60 years. Our finding was similar to that of another study by Gupta et al., where, they reported 93.26% of patients below 60 years of age with chronic NHUs.[9] The most prevalent type of ulcer in our study was traumatic and the patients were improperly treated following trauma. Due to lifestyle-related factors and increased activity level, the middle age group (20–60 years) is most susceptible to trauma. It was seen that diabetic ulcers were more common in the elderly age group.

In our study, 52 (30.9%) patients were either farmers or laborers, constituting the most common occupational group. This finding is in accordance with the finding of Saraf et al.[27] In their study, 42% of individuals were farmers and 16% were skilled and unskilled laborers. Fu et al. also found 37.42% farmers and 26.8% workers in their study.[28] These groups of patients are more vulnerable to trauma. They generally work in barefoot in fields and under harsh climatic conditions. The laborers are at increased risk of fall associated trauma.

We found diabetic foot ulcers were second and vascular ulcers were third common contributors, amounting to 37 (22.02%) and 22 (13.09%) of the NHU cases respectively in our study. In a study conducted by Saraf et al., reported diabetic ulcers as second-most common NHUs in their study, the nonhealing traumatic ulcers were third in order in their study.[27] Gupta et al. also found diabetes (19.35%) as second most common cause in cases of the chronic wound.[9] Chinese hospital-based study has found a low prevalence of vascular ulcers (6.54%) and diabetic ulcers (4.91%) among their patients.[28] Vascular ulcers were the second-most common ulcers in the same study. This observation was different from our study. Vascular ulcers are more prevalent in developed countries possibly due to a sedentary mode of life. Most of our patients were farmers and laborers who have a nonsedentary active lifestyle.

Lower extremities were the most common and head and neck were the least common site of NHU in our study. Our finding was similar to that of Gupta et al. who found 74.2% of chronic ulcers in lower extremities.[9] Fu et al. also found 63.1% ulcers in lower extremities.[28] The factors contributing to ulcers in lower extremities are low socioeconomic status leading to barefoot walking and poor foot care, diabetic ulcers, occupational exposure (farmers and laborers), and poor nutrition. Furthermore, vascular ulcers are predominantly located in the lower extremities.[31]

The prevalence of microbial infections associated with chronic NHUs in this study population was 45.8%. A total of 132 isolates were obtained in our study. Out of the 77 cases, from which microbial agents have been isolated, polymicrobial infections were present in 45 (58.44%) whereas single microbial agents were isolated from 32 (41.56%) cases. Different studies have also shown occurrence of multiple pathogens in chronic NHUs.[10],[12],[15],[17],[18] The number of isolates per ulcer was highest in the diabetic ulcer group (89.19%) followed by traumatic ulcers (87.23%) in our study. A higher percentage of polymicrobial infection was also reported in various other studies.[17],[32] Synergism exhibited by different microbial agents leads to the increased complexity of the infection. Oxygen consumption by aerobic bacteria leads to the development of a hypoxic environment providing an adequate facility for anaerobes to grow. Some anaerobes impair the host immune cells thereby providing an advantage to themselves and to other microbes.[11],[33]

In a recent study by Rahim et al., (2017), common bacterial pathogens associated with chronic wounds were Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia.[10] Bessa et al. in their study, reported the association of bacterial species such as S. aureus, P. aeruginosa, Proteus mirabilis, E. coli, and Corynebacterium spp. in chronic wounds.[16] Gjødsbøl et al. in their study on chronic venous leg ulcers, found anaerobic bacteria in 39.1% of the wounds.[14] Bowler and Davies, found Peptostreptococccus spp, Clostridium spp., pigmented Prevotella and Porphyromonas to be more prevalent in chronic NHUs.[12] In a study conducted by Johnson et al. Peptostreptococci were the most frequently isolated anaerobic bacteria. Peptococcus magnus was the most common species, accounting for half of the total Peptostreptococci.[17]Prevotella, Bacteroides, and Porphyromonas were the other common anaerobes isolated. Sapico et al. found Bactreoides and anaerobic streptococci as the most common bacterial pathogens in their study on 32 patients with chronic ulcers. Clostridium spp. was also a common pathogen found in their study.[18]

In our study, 18 anaerobic isolates were obtained accounting for 13.64% of the total isolates. Most of the anaerobic isolates 8 (44.44%) were obtained from diabetic foot ulcers. 4 (22.22%) anaerobic bacteria were obtained each from vascular and traumatic ulcer cases. Anandi et al., identified anaerobes in 15% of patients of diabetic foot ulcers.[32] In our study, anaerobes were found in 10.71% cases. Anaerobes were associated with 21.62% of diabetic ulcers. The most common anaerobic bacteria isolated in our study were Peptostreptococcus spp. (7.4%) followed by B. fragilis (6.3%). Bowler and Davies found Peptostreptococci as the most common anaerobe in their study. They found particularly Peptostreptococcus spp. and Prevotella spp. to be significantly associated with chronic wounds. However, the B. fragilis isolates in their study were less.[12] Stephens et al. found Peptostreptococcus spp. in more than 75% of chronic wounds they examined.[29] Johnson et al., found, Peptostreptococci as the most frequently recovered anaerobic bacteria.[17] Anandi et al. found the most common anaerobic isolates in their study to be C. perfringens (31%). B. fragilis (20%), C. sporogenes (17.8%), Prevotella spp.(13.3%), and Peptostreptococcus spp. (6.7%) were other isolates in their study.[34] Sapico et al. found Bacteroides as the most common anaerobic bacterial genus in their study.[18] In a study, recent Indian study on surgical infections Antony et al., have reported B. fragilis to be the most common anaerobe involved.[30]

M. chelonae, an atypical, rapidly growing mycobacteria, is associated with chronic NHUs.[35]M. ulcerans, M. marinum, M. immunogenum, M. abscessus, and M. fortuitum are other atypical mycobacteria seen in different parts of the world in association with chronic NHUs.[34] Cutaneous mycobacteriosis usually appears either after contact of (traumatic and surgical) wounds with water or other contaminated products.[19] Atypical mycobacteria are also commonly isolated from chronic ulcers in immunocompromised individuals, especially as a disseminated mycobacterial infection.[34] Reports of immunocompetent hosts getting infected with atypical mycobacteria are also present.[36] The ulcers caused by M. abscessus and M. chelonae are often multiple, whereas those caused by M. fortuitum are single.[36]M. fortuitum is often associated with chronic ulcers caused by trauma and surgical procedure.[25],[36]

Of the 41 isolates obtained from traumatic NHU cases, aerobic bacteria were 29 (70.73%) followed by mycobacteria which were 7 (17.07%). Anaerobes contributed to 4 (9.75%) and fungi to 1 (2.44%). Among the diabetic ulcer isolates (n = 33) aerobic bacteria contributed 20 (60.61%), anaerobic bacteria to 8 (24.24%), and fungal isolates to 5 (15.15%). The highest prevalence of anaerobes was in the diabetic ulcer group. More prevalence of anaerobic bacteria has been shown to be associated with diabetic ulcers by Anandi et al.[32]

Various studies have reported an association of rapidly growing mycobacteria with chronic ulcers. These ulcers have generally been reported following surgical procedures.[36],[37] In our study, out of the eight M. fortuitum isolates, five isolates were obtained as co-infections with aerobic bacteria whereas three were obtained as single microbial isolate causing infection of the ulcers. Out of 11 mycobacterial isolates, 7 (63.34%) were from traumatic ulcer cases. M. fortuitum and M. chelonae have been reported as a cause of abscesses and persistence of infection in surgical sites. They are in all likelihood transmitted by aerosol, dust, or contaminated tap water.[25],[37] The M. chelonae, three isolates of isolates were obtained from patients of chronic osteomyelitis. These patients were not sure about the history of associated trauma. Terry et al., have also reported M. chelonae in patients with chronic osteomyelitis.[35]

Among the fungal pathogens, yeasts are more commonly associated with chronic NHUs than filamentous fungi. Common yeasts isolated are C. parapsilosis, C. tropicalis, Candida guilliermondii, and C. albicans. Among the filamentous fungi Aspergillus, Fusarium, Fonsecaea are more common.[38],[39],[40]

In our study, fungi were isolated in about 5% cases, the most common isolate being C. albicans. In a study done by Bansal et al., on patients with diabetic foot ulcer mixed infection of both bacteria and fungi was found in 8.4%. Fungal isolates accounted for 9% of the total isolates. Yeast were predominant fungi, common isolates being C. tropicalis (29%) and C. albicans (14%). The most common filamentous fungus was Aspergillus flavus.[38] Chellan et al. found 27.2% fungi in their study on type 2 diabetes mellitus patients with chronic skin ulcers. C. parapsilosis was the most common isolate in their study followed by C. tropicalis.[39] A study on 149 nondiabetic individuals with chronic NHUs revealed fungal isolates in 7.4% cases. The most common fungi were C. albicans in the same study.[20] In 11% of the samples in Hansson et al.'s study fungi were isolated in clinically noninfected wounds. F. pedrosoi is prevalent in endemic areas.[40] Cases of F. pedrosoi have been reported in India from patients of NHUs.[41],[42],[43]


As this study was carried out in a tertiary care center, the findings cannot be generalized with other groups of patients, for example, those attending primary care centers. The sample sizes of individual subtypes of ulcers were small, which limits the generalizability of our study. The diagnosis was based on standard laboratory methods and no automation or genetic analysis (e.g. polymerase chain reaction) was used, so the time required for diagnosis was longer.


Polymicrobial infections were seen in more than half of the patients of chronic NHUs. The most common group of microbial agents was aerobic bacteria (S. aureus). There is a need to understand the prevalence and pattern of microbial agents associated with chronic NHU, so that appropriate remedial measures can be taken, which will improve the quality of life of the patient and reduce the burden of care.

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