A Study on Prevalence, Profile, and Risk Factors of Developing Fungal Infection in Patients With Diabetic Foot Ulcer

Diabetes mellitus (DM) is a significant public health challenge worldwide. Foot-related complications of diabetes are more distressing to the patients and health care providers than several other diabetes-related complications.¹ Diabetic foot ulcer (DFU) arises from the interplay of neuropathy and vascular disease in the lower extremities.² Approximately 6.3% of the world population has DFU.² A patient with diabetes has a 15% to 34% lifetime risk of developing DFU.³ A trivial trauma can lead to a chronic foot ulcer owing to several factors, such as sensory neuropathy, impaired healing of the skin, abnormal mechanical loading, and reduced perfusion; conversely, such factors lead to an increased risk of trauma and impaired wound healing.⁴ Patients with diabetes are at 15 to 40 times increased risk of nontraumatic lower limb amputation compared with the general population, and almost 85% amputations in patients with diabetes have DFU as antecedent.⁵ The mortality rate of patients with DFU is twice that of patients with diabetes without DFU.⁶ DFUs are responsible for 8% of all deaths due to diabetes.⁷

The presence of a DFU increases the risk of diabetic foot infection (DFI) and morbidity.^8,9 The global prevalence of DFI is reportedly 25.2% to 58%.⁸ Diabetic foot infection is the most common diabetes-related complication necessitating hospital admission and lower limb amputation. Diabetic foot infections are due to a wide variety of microbial flora. However, the fungal etiology of these infections is not treated with the same regard as the bacterial component.¹⁰ Proper management of DFI includes correct diagnosis, specimen collection for culture and sensitivity testing, use of the correct antimicrobials, and determination of whether surgical intervention is necessary.¹¹ Diabetic foot infections are treated only on the basis of bacterial etiology. However, as untreated fungal infections can be fatal in patients with diabetes,¹² such patients require preventive, diagnostic, and therapeutic interventions for fungal infections.¹³ Owing to a lack of research studies, fungal infections are not usually considered in patients with diabetes; thus, deep wound tissue is not sent for fungal culture and sensitivity testing.¹³ Chronic nonhealing DFUs, which often require amputation, are increasingly associated with fungal infections.¹⁴

The present study evaluated the prevalence, fungal profile, and risk factors of developing fungal infection in patients with DFU to guide the use of appropriate antifungal therapy in order to reduce morbidity and mortality.

Study design. This single-center, prospective observational study was conducted from October 2018 to July 2020 at the Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India. Institutional Human Ethics committee approval and patient informed consent were obtained. All information recorded was kept confidential, and patients were permitted to leave the study at any point without repercussion. All ethical principles included in the Declaration of Helsinki were followed in this study.

Study patients. All patients 18 years and older admitted to the authors’ institution with DFU were included in the study. Patients on antifungal therapy at the time of admission or who received such therapy within 6 weeks prior to admission were excluded from the study.

Sample size calculation. The prevalence of fungal-positive DFUs was used as a primary endpoint for calculating the power of the study. Assuming a proportion of 27.2% fungal-positive DFUs with a precision/absolute error of 5%, power of 80%, and a 10% dropout rate, the final required sample size was calculated to be 304 using the open-source software OpenEpi version 3.01 (Andrew G. Dean, Kevin M. Sullivan, and Roger Mir, Atlanta, Georgia, USA).¹⁵

Sampling technique. All consecutive patients with DFU were taken as study participants.

Study procedure. All consecutive patients admitted to the department of surgery at the tertiary care center with a diagnosis of DFU based on inclusion and exclusion criteria were recruited into the study. Demographic data, comorbidities, and details of DFU were collected, and fasting blood sugar (FBS), postprandial blood sugar (PPBS), and total leukocyte count values were obtained for all participants. Additionally, foot radiography, renal sonography, transcutaneous oxygen pressure (TcPO₂) measurement, the vibration perception threshold (VPT) test, ankle brachial pressure index (ABPI) measurement, and fundal examination were done as part of routine diabetic workup to evaluate complications of diabetes. Foot ulcers were examined and the wound grade (mild, moderate, or severe) documented according to the Infectious Diseases Society of America (IDSA)/International Working Group on the Diabetic Foot (IWGDF) classification.^11,12 After undergoing local examination of the wound, patients underwent surgical debridement either bedside or in the operating room, under local or general anesthesia with adequate aseptic precautions. During this time, 2 exudate samples were collected and sent for fungal culture, bacterial culture, and sensitivity testing. Thorough saline washes were performed after debridement, and 3 deep tissue specimens from the wound bed were collected. Two samples were sent to the microbiology laboratory for fungal and bacterial culture and transported through sterile saline containers. One sample was sent to the pathology laboratory and transported in a formalin container for histopathological identification of fungal elements.
 

Bacterial culture and sensitivity testing. Gram staining was done initially, followed by specimen inoculation on 5% sheep blood agar, MacConkey agar, and brain-heart infusion medium for bacterial culture. The culture mediums were incubated at 37°C for 24 hours, and negative growth mediums were inoculated for another 24 hours before declaring negative growth. Organisms grown in the culture medium were identified by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS; bioMérieux), and conventional biochemical reactions and susceptibility testing were done using the VITEK 2 system (bioMérieux).

Fungal culture and sensitivity testing. Exudates and deep tissue samples were obtained from each patient for fungal microbiological examination; these samples were prepared using 10% potassium hydroxide (KOH) mount for microscopy and fungal culture. The exudate specimen was placed on a clean slide, 10% KOH was added, and the specimen was examined under the microscope. The tissue specimen was cut into smaller pieces (if large in size) and then placed in a test tube with added 10% KOH; the test tube was kept in the incubator at 37°C for 2 hours, after which the specimen was examined under a microscope. The alkali digests the keratin tissue and other tissue materials, enabling the fungal elements to be seen clearly. Next, the examination was done under low power with reduced light for fungal elements. Each sample was aseptically inoculated in Sabouraud dextrose agar for fungal culture in 2 tubes with chloramphenicol. One tube was incubated at 25°C in the biological oxygen demand incubator, and another tube was incubated at 37°C. Sabouraud dextrose agar culture tubes were examined daily for any fungal growth for the first week and weekly thereafter for 4 weeks.

The yeast-like fungal colonies were subjected to Gram staining and MALDI-TOF MS for identification as per manufacturer instructions. The antifungal susceptibility testing of yeasts was done using the aforementioned proprietary system. The filamentous fungal colonies underwent lactophenol cotton blue (LPCB) mount preparation, and the slide culture technique was used for identification. The LPCB stained the filaments and spores light blue and was identified morphologically. Because LPCB mounting sometimes disturbs fungal morphology during the process, slide culture was done to observe the morphology of the fungal isolate and identify it.

Slide culture technique. A corn meal agar block was taken and placed on a sterile slide inside a sterile petri dish, the filamentous fungal isolate was inoculated on 4 corners of the agar block, and a cover slip was placed over it. The whole setup was incubated at 25°C in a biological oxygen demand incubator. After sufficient growth was observed, microscopic examination was carried out by carefully removing the cover slip from the agar block and placing it on another slide with a drop of LPCB for morphologic identification.

Histopathological examination of fungal elements. Microscopic examination to detect fungal elements was done after periodic acid–Schiff stain preparation.

Statistical analysis. The collected data were compiled and then analyzed using SPSS software (version 19.0; IBM Corp.). The results were explained in the form of baseline identification and socioeconomic parameters of the participants in terms of descriptive and inferential statistics. Descriptive statistics included frequencies, proportions, and percentages for categorical variables; continuous variables were analyzed and explained in terms of mean ± standard deviation. The inferential statistics were explained by applying the statistical test to find the significant difference in proportions using the chi-square test. A P value less than .05 was considered significant.

A total of 251 patients were enrolled in the study. Men outnumbered women (76.5% [n = 192] vs 23.5% [n = 59]) (Table 1). The majority of the patients (62.1% [n = 156]) were 51 years or older, and 37.9% (n = 95) were age 50 years or younger. Of the 251 patients, 10% (n = 25) had obesity and 11.6% (n = 29) had DM of 11–15 years. Ninety-seven patients (38.6%) had hypertension.

Based on the clinical characteristics of the DFUs, 53% of patients (n = 133) had mild DFU, 18.7% (n = 47) moderate DFU, and 28.3% (n = 71) severe DFU according to the IDSA/IWGDF classification (Table 2). A significant number of patients (82.8% [n = 208]) had ulcer duration of between 1 and 3 weeks. Sixty-seven patients (26.7%) had foot ulcers associated with osteomyelitis, and 72 (28.7%) had diabetic retinopathy. Twenty patients (8%) had associated peripheral vascular disease, and 70 (27.9%) had diabetic neuropathy. Transcutaneous oxygen pressure was low in 19.5% of patients (n = 49).

Deep tissue cultures were taken from 248 patients owing to logistical reasons. Of these, 18.5% of cases (n = 46) were contaminated. Of the 202 noncontaminated samples, positive fungal growth was observed in 23.3% (n = 47); 2% (n = 4) of the 202 noncontaminated samples exhibited pure fungal growth in the ulcers, and 21.3% (n = 43) had mixed growth with bacteria. Histopathological examination detected no fungal element.

A total of 48 fungal isolates were grown in 47 positive fungal cultures; the prevalence of yeasts was 68.8% (n = 33) and the prevalence of molds was 31.2% (n = 15). The most commonly isolated yeast was Candida tropicalis (27.08% [n = 13]), and C. albicans (20.83% [n = 10]) was the second most common isolate. The prevalence of other fungal isolates was as follows: C. parapsilosis, 8.33% (n = 4); C. auris, 4.17% (n = 2), C. krusei, 2.08% (n = 1); C. glabrata, 2.08% (n = 1); C. orthopsilosis, 2.08% (n = 1); and Trichosporon species, 2.08% (n = 1). The most common molds grown were Aspergillus species (10.41% [n = 5]) and Fusarium species (10.41% [n = 5]), followed by Rhizopus species (4.16% [n = 2]), Penicillium species (4.16% [n = 2]), and Cladosporium species 2.08% (n = 1) (Figure 1). Of the 47 patients with positive fungal growth, 91.5% (n = 43) had both bacterial and fungal growth positivity, whereas 8.5% (n = 4) had fungal growth only. Figure 2 demonstrates the resistant pattern of yeasts to individual antifungal drugs.

No statistically significant association was found between presence of fungal growth and age, sex, BMI, or chronicity of DM. Among 47 patients with positive fungal growth, 61.7% (n = 29) had an FBS level above 125 mg/dL and 38.3% (n = 18) had an FBS level at or below 125 mg/dL. PPBS value was within the range of 140 to 200 mg/dL in 48.9% of patients (n = 23) and was greater than 200 mg/dL in 46.8% (n = 22). Only 4.3% patients (n = 2) had a PPBS value of less than 140 mg/dL. No statistically significant association was found (Table 3).

A significant association was found between wound grade (P = .027), ulcer duration (P = .028), and positive fungal growth in DFUs. However, no statistically significant association was found between fungal growth and the DFU complications of osteomyelitis, neuropathy, peripheral arterial disease, or low TcPO₂ levels (Table 4).

The present study showed a 23.3% prevalence of fungal infection in DFUs, with C. tropicalis (27.08%) the most common isolate; however, 23% of C. tropicalis cases were resistant to fluconazole. Patients with mild DFU and ulcer duration of 7 to 14 days had a high rate of fungal infections, but no significant association was found between fungal growth and other risk factors.

Of the age groups studied, patients aged 51 to 60 years comprised the largest group (31.5%). This finding is comparable to that of previous studies.^16,17 This evidence shows that the risk of DFU and DFI increases with age. Kalan et al¹⁸ reported that the proportion of DFUs was higher in males than females (78% and 22%, respectively), which is comparable to the present study (76.5% and 23.5%, respectively). In the present study, 45.1% of patients had an increased BMI (ie, overweight, obese), compared with 64.3% in the study by Sohn et al.¹⁹ A possible explanation for normal BMI in 46.2% of patients in the present study could be a catabolic response to the nonhealing DFU and associated inflammation. In the present study, 46.2% of patients had a history of diabetes of less than 5 years’ duration, whereas Heald et al²⁰ reported patients with a long-standing history of diabetes for around 11 years. Possible reasons for the shorter time from diabetes onset to DFU in the present study could be poor glycemic control and the habit of barefoot walking. However, previous studies have found a correlation between poor glycemic control and fungal infection in DFU.^21,22

In the present study, most patients (53%) had a mild wound according to the IDSA/IWGDF classification, whereas patients in previous studies had higher Wagner wound classification grades.^23,24 Early presentation to the hospital may be the reason that the majority of ulcers in the present study were mild. In the present study, the prevalence of fungal infection was 23.3%, with 13 different fungal isolates. Previous studies showed a higher prevalence of fungal infection (80% vs 100%) because of the selective recruitment of patients highly suspicious for fungal infection and those with fungal hyphae on routine microscopy.^18,20

C. tropicalis was the most commonly isolated fungus (27.08%) in the present study; this species was also the most commonly isolated fungus in a study done in India by Sanniyasi et al.¹⁴Aspergillus species and Fusarium species were the most commonly isolated molds in the present study, a finding similar to that of previous studies.^18,21 In the present study, 18.5% of specimens sent for fungal culture sensitivity testing were contaminated. Contamination might have been caused by bacterial overgrowth despite the use of chloramphenicol while inoculating samples for fungal culture because of its limited antibacterial spectrum. The novel Cabin-Sequestering method for removing bacteria from fungi cultured on a solid medium can be used to reduce contamination.²⁵

In the present study, almost every case of C. albicans, C. glabrata, C. orthopsilosis, and C. parapsilosis grown in deep tissue cultures was sensitive to fluconazole, voriconazole, or amphotericin B. However, 23% of C. tropicalis cases were resistant to fluconazole and 7.69% were resistant to voriconazole and amphotericin B. In a study done in India, Chellan et al²¹ found that 13.3% of C. albicans isolates and 9.3% of C. tropicalis isolates were resistant to fluconazole, which is comparable to the findings in the present study.

In the present study, 91.5% of fungal isolates had coexistence of bacterial growth. However, in a study conducted in Croatia, Missoni et al²⁶ reported that 68.2% of fungal isolates had bacterial coexistence. The prevalence of bacterial and fungal coexistence was high in the present study because the deep tissue specimens were sent before antibiotics were started. Additionally, fungal elements were not detected on histopathological examination, possibly owing to superficial fungal infections in the ulcers. Previous studies reported fungal positivity in patients with ulcer duration between 8 and 11 weeks.^18,26 In the present study, there was a significant association (P = .028) between ulcer duration and fungal growth positivity in tissue culture; 51.1% of patients with positive fungal growth had ulcer duration between 7 and 14 days. A possible explanation for this might be the institution of empirical antifungal therapy or the colonizing nature of most of the fungi isolated. A significant association (P = .027) was found between wound grade and fungal culture positivity; 38.3% of patients with fungal culture positivity had severe DFU based on the IDSA/IWGDF classification. This might be due to fungi's invasive nature, which could have elicited a systemic inflammatory response syndrome, although blood culture was not sent for the study participants. No statistically significant association was observed in the current study between fungal infection and a patient’s age, sex, duration of diabetes, blood sugar level, ABPI, VPT, TcPO₂, or presence of osteomyelitis, which is similar to findings observed in other studies.^13,18,21

Patients with chronic nonhealing wounds despite proper antibiotic therapy and the presence of fungal hyphae on routine microscopy are at high risk for fungal infection.²⁰ It is also known that fungal detection rate varies in different evaluation methods (5% by routine microscopy vs. 80% by PCR).¹⁸ The present study indicates the need for fungal evaluation of chronic nonhealing DFUs with rapid progression despite antibacterial therapy and wound care, because the introduction of antifungal treatment results in a faster wound healing rate with reduced risk of amputation. The authors of the present study believe these results will alert clinicians of the need to treat fungal infections and encourage further research. Further randomized controlled trials comparing the treatment groups with and without antifungal therapy are needed.

This study has limitations. It was conducted at a single center, and only 251 patients were included owing to logistical reasons. Immunosuppressive therapy and neutropenia were not included in the exclusion criteria. Hemoglobin A1c was used as the indicator of glycemic control for the 3 months prior to admission, and it was not routinely evaluated for all participants. A significant number of specimens for fungal culture were contaminated, which might decrease the yield of fungal growth.

This single-center, prospective observational study evaluated the prevalence, profile, and risk factors of developing fungal infection among 251 patients with DFUs with the aim of guiding the use of antifungals in the management of DFIs. Subgroup analysis showed a 23.3% prevalence of fungal infection in DFUs, with C. tropicalis the most common isolate. Approximately 23% of C. tropicalis cases were resistant to fluconazole. Patients with mild DFU according to the IDSA/IWGDF classification and ulcer duration of 7 to 14 days had a high rate of fungal infections. However, no significant association was found between fungal growth and blood sugar level, duration of diabetes, osteomyelitis, neuropathy, or vasculopathy. Further studies are needed to assess the importance of fungi in DFUs and antifungals in managing DFIs to reduce the morbidity and mortality associated with fungal infection in DFUs.

¹Department of Surgery, ²Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India 605006

Gopal Balasubramanian, MS; Additional Professor, Department of Surgery, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India 605006; drbala18@gmail.com

No authors have any conflicts of interest. No financial support was given for this work.

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