Journal of Dr. NTR University of Health Sciences

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 10  |  Issue : 2  |  Page : 88--93

A study of evaluation of wound swab for bacteriological culture and antibiotic sensitivity in comparison with tissue sample culture in diabetic patients with infected foot ulcer


Akshath Gurulingaiah, Alok Bhalla, Simarjit S Rehsi, Pankaj P Rao 
 Department of Surgery, Armed Forces Medical College, Pune, Maharashtra, India

Correspondence Address:
Dr. Simarjit S Rehsi
Department of Surgery, AFMC, Pune - 411 040, Maharashtra
India

Abstract

Background: Most diabetic wounds are infected and eradication of the infected source is paramount to the success of healing. Antibiotic culture and sensitivity of samples aid in the assessment of the severity of the infection and the state of sensitivity or resistance of organisms in an infected ulcer to antibiotic treatment, thereby preventing or minimizing the necessity of amputation. Aims: To compare two methods of evaluating bacterial infection, wound swab culture (SC) against tissue sample cultures (TC), and determine sensitivity patterns and compare them for establishing the diagnosis of infected diabetic foot ulcers. Methods and Material: New-onset diabetic foot ulcers were subjected to tissue sampling and wound swabbing; specimens were cultured for aerobic and anaerobic organisms. a. Settings and Design: Prospective observational study at a tertiary care hospital done over two years. b. Statistical analysis used: Data was recorded in excel data sheet and statistically analysed by using the SPSS Ver 22.0. Results: Three hundred and eight micro-organisms were isolated from a cumulative total of 127 infected wounds (2.75 and 2.08 per wound for SC and TC, respectively). Sensitivity and specificity were 82.27% vs 78.48% and 71.43% vs 52.38% for the SC and TC groups, respectively. The maximum concordance was noted in the Grade 2 ulcer group (73.84%). Conclusion: Wound SCs were comparable to TCs for Grade 2 ulcers. For higher grades of ulcers, tissue sampling method is recommended.



How to cite this article:
Gurulingaiah A, Bhalla A, Rehsi SS, Rao PP. A study of evaluation of wound swab for bacteriological culture and antibiotic sensitivity in comparison with tissue sample culture in diabetic patients with infected foot ulcer.J NTR Univ Health Sci 2021;10:88-93


How to cite this URL:
Gurulingaiah A, Bhalla A, Rehsi SS, Rao PP. A study of evaluation of wound swab for bacteriological culture and antibiotic sensitivity in comparison with tissue sample culture in diabetic patients with infected foot ulcer. J NTR Univ Health Sci [serial online] 2021 [cited 2022 Jan 24 ];10:88-93
Available from: https://www.jdrntruhs.org/text.asp?2021/10/2/88/332852


Full Text



 Introduction



Diabetes mellitus has attained a worldwide pandemic status in recent decades with its prevalence in the world exceeding in significant proportions. India, also with its approximately 42 million cases, is ranked first in the list of the ten nations most affected with diabetes.[1] Foot ulcerations are commonly related to peripheral sensory and motor neuropathy and peripheral arterial disease[2] and occurs in 15%–25% of the patients during their lifetime.[3] More than 50% of patients presenting with diabetic foot ulcer have concomitant infections,[4] and foot infection precedes approximately 80% of nontraumatic lower-limb amputations.

Most diabetic wounds are infected and eradication of the infected source is paramount to the success of healing. They are polymicrobial in nature with Staphylococci being the most common organisms. For detection of infection, a postdebridement specimen is an important tool for aerobic and anaerobic culture. Broad-spectrum antibiotic coverage is required for chronic and severe infections. The cause of persistent cellulitis and nonhealing infection is usually an underlying deep infection or osteomyelitis. Treating these wounds is a constant challenge for the surgeon. Failure to resolve the cellulitis over several days indicates the inadequacy of antibiotic coverage, and it needs to be re-evaluated. Tissue samples and wound swabs for antibiotic culture and sensitivity aid in the assessment of the severity of the infection and the state of sensitivity or resistance of organisms in an infected ulcer to antibiotic treatment, thereby preventing or minimizing the necessity of amputation in diabetic patients with an infected foot ulcer.

This study was performed to compare two methods of evaluating bacterial infection, wound swab culture (SC) against tissue sample cultures (TC), to determine sensitivity patterns, and compare them for establishing the diagnosis of infected diabetic foot ulcers.

 Methods



This cross-section observational study was conducted in a tertiary care teaching hospital for a period of 2 years from August 2018 to August 2020. All in-patients and out-patients with infected diabetic foot ulcer and not on any systemic antibiotics for the past 4 weeks were recruited in the study after written informed consent. Permission for the study was granted from the Institutional Ethics Committee.

Patients with immunocompromised state other than diabetes, creatinine >2 mg/dL, albumin <3 g/dL, hemoglobin <9 g/dL, those already operated upon with stump ulcers, patients with gangrene/absent pulses, or h/o stroke were excluded from the study.

The patients with new-onset diabetic foot ulcers were screened for eligibility, informed about the trial, and included in the study after consent. These patients were subjected to both swab and deep tissue cultures. The wound was cleaned with copious amounts of saline and gauze, and debrided of the necrotic material, foreign particles, calluses, and undermined wound edges. After cleaning and debridement, two cultures were simultaneously taken from each patient by swabbing and deep tissue culture. No antimicrobial agents such as alcohol or iodine or any antiseptic were used for the wound cleaning before specimen collection. Only one site was sampled from each patient.

Superficial wound SCs were obtained using the Levine technique, where a wound swab was rotated over an area of 1 cm of the wound for 5 s, using sufficient pressure to extract fluid or pus from the inner parts of the wound.

TC samples of about 4 mm in diameter were collected from the junction of non-viable and viable tissue by the use of artery forceps. All nonviable tissue was removed from the wounds, and extension of any sinus tract or abscess performed in the deep tissue debridement was collected for TC. The samples obtained were sent to the laboratory by inserting those into a transport tube containing brain infusion broth suitable for both aerobic and anaerobic micro-organisms within 15 min after collection for immediate processing in the laboratory. The culture of aerobic and anaerobic species was done by inoculating on to the blood agar, Eosin-Metilen Blue (EMB) agar, Sabouraud agar, and Wilkins-Chagren anaerobe agar at 35–37°C for 24–48 h. The catalase test, hemolysis reaction, optochin, bacitracin, and cotrimoxazole susceptibility testing were performed for the Gram-positive bacteria. The oxidase test was applied for Gram-negative bacteria. The Kirby-Bauer Disc diffusion sensitivity testing was also performed for the optimization of antibiotics for the treatment of the patient.

The patients were given empirical broad-spectrum antibiotics initially along with appropriate wound care management. Sterile dressings were applied daily following required wound debridement.

In this study of 121 patients, the results of the cultures were available by the Day 05;, therefore, the antibiotics were changed according to the results of antibiotic sensitivity. The results of the first culture samples were evaluated for the study. The subsequent cultures were sent for persisting wound infections on Day 11 and Day 21 of the admission. The sensitivity patterns were used to provide appropriate antibiotics for subsequent management. However, the results of these cultures were beyond the scope of the present study and, thus, were not used. The patients with Grade 4 ulcer, mostly having underlying abscess, were treated under intensive care with proper wound debridement and higher antibiotics.

Data analysis

All the data generated were recorded in an excel datasheet and statistically analyzed by using the SPSS Ver 22.0 (IBM Corporation, USA) for MS Windows. The nonparametric variables were depicted as either/or values. The categorical variables were presented as absolute values or percentage/proportions; numerical variables were expressed as mean ± standard deviation (SD) or median (range) as per data distribution pattern.

The categorization of patients as infected/non-infected was decided on the Infectious Disease Society of America (IDSA)/International Working Group on the Diabetic Foot (IWGDF) CLASSIFICATION with relevant cultures and accordingly, the patients were categorized into true positive (TP), false positive (FP), true negative (TN), and false negative (FN) in both the groups. The two-sample t-test (Student's t-test) was used for analyzing the quantitative variables with normal distribution. The Chi square (χ2) test was used where distribution was skewed and for categorical variables. All the hypotheses were formulated using two-tailed alternatives against each null hypothesis (hypothesis of no difference). P values less than 0.05 were statistically significant. In comparisons using the Student's t-test, 80% confidence intervals for the mean difference in response provided a range of likely values to assess clinical significance.

 Results



In this study, 157 patients were evaluated, of which 133 patients were eligible for the study. The diabetic foot ulcer (DFU) wounds of these patients were processed for both TC and SC. Four patients were lost to follow-up, three patients refused to consent, and five patients underwent amputation due to severe sepsis. The total number of patients was 121 out of which 94 were male, and the rest were female. Most of the patients lied in the age group of >50 with maximum patients in the age group of 51–60 years for both genders. There was no significant difference with the P value of 0.99 [Table 1].{Table 1}

According to the IDSA classification, patients were classified into four grades with 42, 25, 41, and 13 patients in Grade 1 to 4, respectively. Three hundred and eight micro-organisms were isolated from both the swab and tissue specimens from a cumulative total of 127 infected wounds (average organisms – 2.75 and 2.08 per wound for SC and TC, respectively). In SC and TC groups, 20 and 12 wounds, respectively, grew Gram-positive organisms which were discounted from results as likely contaminants. There was a trend for positive cultures for Gram-positive microbes in Grade 2 and 3 ulcers in both TC and SC. However, this bias reversed towards Gram-negative organisms in Grade 4 ulcers. The number of patients from which organisms were isolated in the SC group was 65 out of a total of 121 patients (53.72%), and for the TC group it was 62 patients out of a total of 121 (51.24%). The most prevalent Gram-positive organisms in both SCs and TCs were Staphylococcus aureus (17.53%) and Coagulase-Negative Staphylococci (CONS) (12.66%); in Gram-negative these were E coli and Klebsiella sp. Even though comparable numbers of Gram-negative organisms were isolated from both groups, isolation of Gram-positive microbes was much more in the SC group. Anaerobes were predominantly grown from deep TCs [Table 2].{Table 2}

The sensitivities of both the groups were comparable at 82.27% and 78.48% for the SC and TC groups, respectively. In contrast, the TC group had much higher specificity at 71.43% in comparison to 52.38% for the SC group [Table 3], [Table 4] and [Figure 1].{Table 3}{Table 4}{Figure 1}

The maximum concordance was noted in the Grade 2 group with 48 wounds out of a total of 65 with the same bacteriological profile. This was substantially lower in both Grade 3 and 4 groups with 23 and 20 samples showing concordance [Figure 2].{Figure 2}

 Discussion



Infections have been innately associated with diabetic foot ulcers due to varied reasons. Early recognition and management of diabetic infections are, therefore, associated with reduced morbidity and significantly reduced chances of limb amputation. Moreover, the consequent reduction of hospital stay also results in lower economic costs to the patients and Medicare system. A good quality specimen for culture from clinically infected wounds helps in the appropriate selection of antibiotic regimes and alteration of empirical antibiotics already employed by the clinicians.[5] There has been an ongoing debate about the reliability of different sampling methods for the microbiological assessment of infected diabetic foot ulcers. Lately, there have been improved outcomes of cultures even in superficial swab methods due to much improved surgical methods for collection of specimens including Levine's technique, needle aspiration, and tissue biopsy. Superficial swabbing also carries the advantage of lower risk of introduction of infection into the wounds.

As per the current recommendations, the standard for microbiological diagnosis and identification of the causative organism is a quantitative tissue biopsy of the diabetic ulcer wound.[6] A quantitative biopsy is a time-consuming method used in research settings as it requires quantification of bacterial burden per gram of tissue at a level of ≥105 CFU/g of tissue. Moreover, it is expensive, generally not available, and requires a higher skill level and experience and, thus, not used routinely in clinical settings.[7]

Obtaining superficial SCs is relatively easy and can be done even by semi-skilled workers. They are cheaper to collect by a margin and do not require any previous experience. However, this relative ease of collection leads to several disadvantages of collecting superficial bacterial contaminants of skin, especially when employed over uncleaned skin. And as an obvious corollary, it cannott be used for obligate anaerobes and fastidious micro-organisms. The collection of contaminants can be decreased to an extent with proper cleaning of the wound before swabbing and using special methods such as Levine's technique.

Hence, if the superficial SC method is proven comparable to the deep TCs, it can potentially provide an easier and less complicated means of diagnosing diabetic foot infection. It will be less traumatic for the patient under treatment too. The collection of deep TCs nowadays is obtained by curettage of the base of a debrided ulcer, aspiration of any purulent secretions, or procuring a tissue biopsy.[8],[9]

In the present study, a total of 308 micro-organisms were isolated from both the swab and tissue specimens from a total cumulative of 127 infected wounds. A mean of 2.75 species per wound for swabbing and 2.08 species per wound for TC groups were isolated. The study by G. Pellizzer et al.[10] also reported similar numbers of 2.34 species per patient by swabbing and of 2.07 by TC methods. Similarly, others[11–13] have also reported numbers between 1.6 and 2.06 isolates per patient. In addition to these reported incidences, few other authors[14],[15] report isolates of four or more species per patient. The reasons for this discordance in reported numbers can be due to the method of collection of specimens, the cleaning techniques before sampling, and regional variations in addition to the selection of severity patients for the individual studies. Contrary to these figures, Kessler et al. observed that the number of micro-organisms isolated by swabbing to be lesser as compared to the deep TC method at 1.09 vs. 2.04 (P < 0.02).[16]

The Gram-positive cocci were the commonest organism found in our study. Staphylococcus aureus was isolated most frequently followed by CONS. This was broadly in line with the findings of many other studies.[17–19] This finding is especially true for the studies where patients of diabetic foot infection have been studied who have not been exposed to any antibiotics.[20],[21] The prevalence of typical Gram-negative bacteria, especially E coli and Klebsiella sp. were next most common. In recent studies, Pseudomonas has emerged as an upcoming pathogen in increased numbers, more so in studies done in warmer climates and in patients exposed to antibiotics.[22] Thus, it has been recommended to also include anti-pseudomonal antibiotics in the antibiotic cocktail to treat patients of infected diabetic foot.

Sapico et al.[14] had observed no significant differences when comparing the result of sampling by swabbing, curettage of the ulcer base, and needle aspiration with deep tissue culturing taken as a reference method.

Another retrospective study[13] reported that cultures from curettage of the deepest site of the ulcer had a yield similar to deep intraoperative culture collection. Adequate isolation of both aerobic and anaerobic microbes is feasible even by surface swabbing if appropriate microbiological culturing techniques are used.[23]

Contrary to the conventional wisdom that superficial swabs are not ideal for culturing, our study showed marginally better sensitivity with superficial swabbing as compared to deep TCs. Provided that specimen handling is adequate and laboratory processing prompt enough, our study suggests that both superficial swabbing and deep TC can yield similar results at the onset of starting therapy, both in terms of isolates per patient and the pathogen isolated. This concordance was maximum in Grade 2 wounds which showed a rate approaching three-fourths of the total specimens. This finding was comparable to the study done by Huang et al.[24] who reported a similar concordance rate of 80%.

Thus, we can recommend superficial swabbing as a method of specimen collection for cultures in moderate wounds. Deep wounds and osteomyelitis will require proper debridement and collection of deep tissue or bone fragments for the identification of pathogens.

The major limitation of our study was that it was done in a single center with limited patients due to constraints of time and COVID-19 epidemic. Moreover, fungal cultures were not performed which can potentially alter the results. Fungal infections are being increasingly recognized, especially in immunocompromised patients of diabetes mellitus with chronic wounds and ulcers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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