Associations Among Wound-Related Factors Including Biofilm,Wound-Related Symptoms and Systemic Inflammation in Older Adults with Chronic Venous Leg Ulcers

Abstract

Objective:

The purposes of this observational prospective study were to (1) characterize the wound-related factors (wound area, the presence of biofilm, and total bacteria), wound-related symptoms (fatigue, pain, exudate, itching, and edema or swelling), and systemic inflammation (level of serum C-reactive protein [CRP]), and (2) explore associations between wound-related factors, wound-related symptoms, and systemic inflammation in older individuals with chronic venous leg ulcers (CVLUs) over 8 weeks of wound treatment.

Approach:

A total of 117 participants who received standardized care (weekly sharp debridement) for chronic venous ulcer were enrolled. We collected clinical data every 2 weeks during the 8 weeks of the study period or until the wound was healed (if healed before 8 weeks). Associations among variables were estimated using a Bayesian approach applied to general linear mixed models.

Results:

Based on Bayes factor (BF) value, there was extremely strong evidence for the association of biofilm with mean total bacteria (BF >1,000). There was moderate evidence of a direct association between biofilm presence and levels of CRP (BF 4.3) and moderate evidence of direct associations between biofilm and wound-related symptoms, pain and exudate (BF 5.12, 8.49, respectively).

Innovation:

Wound-related symptoms and the level of systemic CRP were associated with biofilm among patients who were receiving weekly sharp debridement. Symptom severity associated with CVLUs requires assessment and management of wound-related factors and levels of inflammation in addition to symptom assessment.

Conclusion:

This study is the first to examine associations among biofilm, as wound-related factors, systemic inflammation, wound-related symptoms, and wound healing in clinical settings. Symptom severity, level of systemic CRP, and wound-related factors should be considered as well as assessment of biofilm in CVLU in older individuals with CVLU.

Debra Lyon, PhD

INTRODUCTION

In 2014, about 8.2 million people had chronic wounds and the treatment costs of chronic wounds ranged from $28.1 to 96.8 billion in the United States.^1,2 The numbers have increased: in 2019, 10.5 million Medicare beneficiaries received treatment for chronic wounds.^3,4 Since venous leg ulcers (VLUs) tend to be chronic owing to their high susceptibility to infection and high recurrence rate,^5,6 they account for the majority of chronic wounds. Patients with VLUs suffer from diverse symptoms, including pain, fatigue, depression, swelling, and exudate,^7,8 and most patients with VLUs who have delayed healing experience significant symptoms.⁹ Chronic wounds are associated with comorbidity, wound inflammation, aging, and negative emotion.^10

–13 Perturbances in the inflammatory response may contribute to both the wound healing trajectory and the development and severity of symptoms.

Wound-related factors may contribute to symptoms in individuals with chronic venous leg ulcer (CVLU). Biofilms consist of aggregates of microbial cells embedded in an extracellular polymeric matrix and often attached to living or nonliving surfaces.¹⁴ Growth within a biofilm provides microorganisms with protection from immune responses and antimicrobial agents.¹⁵ Biofilms are believed to delay wound healing, although there has been lack of data from randomized controlled human clinical trials.¹⁶ Potential mechanisms of biofilm-impaired wound healing have been investigated using animal models. Biofilm-impaired reepithelialization has been reported from studies with mouse models using both Staphylococcus aureus ¹⁷ and Pseudomonas aeruginosa ^18,19 and a rabbit ear model using S. aureus.²⁰ The latter study also compared cytokine (IL-1α and TNF-β) expression between planktonic and biofilm infections, which indicated a lower inflammatory response to biofilm infection. Indeed, the presence of planktonic and biofilm microorganisms modify immune responses in acute and chronic wounds.¹⁴ According to a recent review, chronic wound infections are characterized by microbial-controlled inflammatory responses rather than host-controlled immune responses.²¹

Although much insight has been gained from rodent and rabbit models, there are important differences compared with human wound healing. Porcine models better represent human wound healing^22,23 and have demonstrated biofilm formation²² and biofilm-impaired skin barrier function.²⁴ Although animal studies have provided important insights into biofilm-associated wound repair defects and inflammatory responses, it is important to note that animal studies alone cannot fully explain the complex interactions between biofilms, inflammation, and wound healing in the clinical setting, as they cannot take into account the various physiological, psychological, and social factors involved in human wound healing.

When wounds become infected, affected individuals may manifest signs of infection such as pain, erythema, tenderness, and swelling,^25,26 although these signs may be less pronounced in chronic biofilm infections than acute planktonic infections.¹⁷ There is growing recognition that wound-related symptoms are common in both acute and chronic wounds and impact the quality of life in individuals with chronic wounds. Wound-related symptoms experienced by patients with CVLU have a negative impact on quality of life.^9,27 These symptoms may be associated with the severity of wound conditions, including the presence of biofilm, which reflect the wound healing trajectory. However, there is little research in humans, which examine these relationships. From a symptom science standpoint, associations between biofilm, inflammatory response, wound-related symptoms, and wound healing in clinical settings have not been clearly described.

Therefore, by examining wound-related symptoms and examining the relationships with biofilm and inflammatory markers such as C-reactive protein (CRP) during the course of wound treatments in human studies, clinicians may develop interventions to not only promote wound healing but also decrease symptom burden, thereby improving the quality of life in individuals with CVLU. The purpose of this observational prospective study was (1) to characterize the wound-related factors (wound area, the presence of biofilm, and total bacteria), wound-related symptoms (fatigue, pain, exudate, itching, and edema or swelling), and systemic inflammation (level of serum CRP) and (2) to explore associations between wound-related factors, wound-related symptoms, and systemic inflammation in individuals with CVLU over 8 weeks of wound treatment. To accomplished the objectives of the study, we examined the presence of biofilm from debrided wound tissue, serum CRP, wound-related symptoms (fatigue, pain, itching, exudate, and edema or swelling), and healing outcome (wound area) over 8 weeks for each subject. In addition, we described associations between wound-related symptoms, CRP, and wound area with biofilm scores.

CLINICAL PROBLEM ADDRESSED

About 2% of people experience VLUs, and up to 5% of the elderly are affected.^28,29 Owing to slow healing and the recurrent nature of VLUs,^30,31 the socioeconomic impacts of VLUs are tremendous, including hindering occupational activities, worsening quality of life, and financial burden up to 2.5 billion annually for treatments.^10,32 Indeed, the individual treatment cost for a person with venous ulcers is estimated to be $10,560–$34,000 annually.³³

Although treatment guidelines for VLUs (e.g., compression, dressing, pentoxifylline, knee stocking, and surgery) are well established, diagnostic and prognostic measurements are not yet clearly established.³¹ To provide optimal treatment for wound healing, prognostic factors that contribute to wound healing trajectory is essential. Describing the change of wound-related symptoms and systemic inflammatory markers may also give clues that support the prediction of wound healing. As far as we know, this is the first longitudinal study that characterizes biofilm and wound-related symptoms under weekly sharp debridement in individuals with CVLU.

MATERIALS AND METHODS

Study design

This study was an observational prospective study supported by the National Institute for Nursing Research (NINR) (RO1 NR016986).

Recruitment of subjects

This study was approved by the institutional review board of the University of Florida (IRB201700566). Participants were enrolled in the study after signing the written consent form. A total of 117 subjects who received weekly sharp debridement at a wound clinic were enrolled in the study. Inclusion criteria were the following: Subjects were (1) aged 55 years or older; (2) had a VLU confirmed by clinical diagnosis; (3) had an adequate arterial blood perfusion (ankle brachial index between 0.7 and 1.3, inclusive or no occlusion as determined by Doppler measurement); (4) had a chronic venous ulceration duration of >30 days; (5) were cognitively intact as determined by a minimum score of 24 on the Mini-Mental State Examination.

Exclusion criteria were the following: subjects (1) undergoing kidney dialysis for renal failure; (2) receiving immunosuppressant treatment, including systemic steroids or topical steroids in the wound within 4 weeks before the study; (3) had a systemic infection; (4) received chemotherapy within 4 weeks before study entry; (5) had a severe concomitant condition; (6) had immune suppression (HIV, transplant status) or auto immune disorders. Detailed inclusion and exclusion criteria are described elsewhere.^34,35

Data collection

Data were collected at baseline and biweekly (2, 4, 6, and 8 weeks) of treatment or until the wound was healed (if healed before 8 weeks) between August 2018 and June 2022. Each patient received follow-up care during the treatment period with the same physician, using a standard protocol.

Participants' characteristics

Demographic characteristics were collected from electronic health records or a demographic questionnaire that included age, gender, race, ethnicity, educational level, marital status, and economic status. Body mass index (BMI) and Charlson comorbidity index (CCI) were also calculated, and collection methods are described elsewhere.³⁴ Use of antibiotics and oral pain medications were retrieved from the electronic health record. Healed participants refer to those whose wounds were identified as clinically healed by the provider at some point during the 8 weeks, whereas nonhealed participants had wounds not declared as clinically healed during the 8 weeks. All decisions regarding wound healing status were determined by physician's clinical judgment and recorded in the medical record. Participating physicians were wound specialists who were working in the wound care clinic and following a standardized protocol. None of the subjects were treated with compression modalities during the course of the study.

Wound symptoms: pain, exudate, itching, and edema or swelling

The Toronto Symptom Assessment System for Wounds was used to measure wound-related symptoms, including pain, exudation, and edema or swelling. This measure assesses these nine dimensions of wound-related pain and discomfort using a 0 (none) to 10 (most severe) scale.³⁶ We used the pain, exudate, itching, and edema or swelling scales in this study.

Fatigue

Fatigue was measured by the Brief Fatigue Inventory (BFI), which has a 9-item, 11-point rating scale (0 indicates no fatigue and 10 indicates most severe fatigue).³⁷ The BFI is a clinically validated tool used to assess this common and debilitating symptom in adults experiencing a chronic condition. On the BFI, severe fatigue can be defined as a score of 7 or higher. The BFI has demonstrated excellent reliability in clinical trials, with Cronbach's alpha ranging from 0.82 to 0.97.³⁷

Serum CRP

CRP is a clinically validated marker of inflammation. Measuring CRP values can prove useful in determining the degree of systemic inflammation as well as progress or the effectiveness of treatments. Normal CRP concentration in healthy human serum is usually <10 mg/L, slightly increasing with aging. Higher levels are found in mild inflammation and viral infections (10–40 mg/L), active inflammation, bacterial infection (40–200 mg/L), and severe bacterial infections. Serum CRP was measured using enzyme-linked immunosorbent assay (ELISA), a commercial assay, according to manufacturer's instructions (DuoSet^® ELISA Development System, human C-reactive protein; R&D Systems, Minneapolis, MN).

Procedure of wound tissue acquisition

The physician performed wound tissue acquisition during the sharp debridement procedure. The physician used a dermal curette and/or scissors, forceps or a scalpel as needed. The debridement of the edges of wounds was performed first followed by the base of the wound. The pattern of debridement thereafter varied based on what was judged most appropriate to accomplish wound healing. The physician debrided in layers until obtaining viable tissues because the VLUs were typically quite exudative. If a patient had multiple wounds, tissue was obtained from the largest wound.

Biofilm scores

The wound tissue samples were embedded in OCT (Tissue-Tek^® Optimum Cutting Temperature; Sakura Finetek) and frozen. Frozen samples were cut into 5-mm thick sections using a cryostat, placed on plus slides and stored at −70°C.

General biofilm analysis

The specimens were stained using Sytox^® green and wheat germ agglutinin conjugated with Texas Red^® (WGR-TR; Life Technologies, Carlsbad, CA), and examined using a Leica SP5 confocal scanning laser microscope. Specimens were scored for biofilms using a 6-point scale; 0 = no bacteria observed, 1 = single individual microorganisms, 2 = small microcolonies (10–100 cells), 3 = large microcolonies (>100 cells), 4 = continuous biofilm, and 5 = thick (>10 μm) continuous biofilm.³⁸ In this study, we treated biofilm as a binary variable, whether biofilms were present (biofilm score of 2 or greater) or not (biofilm not present was the referent category).

Quantitative functional biofilm analysis of the total bacteria

The levels of total bacteria were measured using minor modifications of the basic technique that was described by Phillips et al.³⁹ In brief, the curettage samples harvested from patients were immediately placed in a sterile 15 mL tube with 1.5 mL phosphate-buffered saline containing 5 ppm Tween-20 and transported to the laboratory at 4°C using cold packs. After measuring the sample weight (mg) using a microbalance, samples were vortexed for 1 min at high speed, which dislodges both planktonic and biofilm bacteria from the curettage tissue sample. The level of total bacteria was measured in an aliquot (400 μL) of the vortexed sample that is subjected to five cycles of ultrasonic treatment to disperse the biofilm components into single cells, followed by dilution plating onto a nonselective Tryptic Soy Agar plate.

After incubation for 48 h at 32°C, the number of colonies were counted on the dilution plates that contained between 15 and 300 colony-forming units (CFU), or 0 CFU if no colonies were present on the 10-fold dilution plates. Values of total bacteria were expressed as CFU/g of curettage sample.

Statistical analysis

A total of 117 participants were included in the analysis. Distributions for each variable were examined with descriptive statistics appropriate for measurement level. Owing to the skewed distributions of the wound area and total bacteria variables, a log base 10 transform was applied to normalize the distribution of values. Patterns of missingness were evaluated using the nonparametric omnibus test for missing completely at random (MCAR) as implemented by TestMCARNormality within the MissMech R package.^40

–43

Separate generalized linear mixed models, one for each of the outcome variables (wound area, pain, fatigue, exudate, itching and edema or swelling, serum CRP and total bacteria), and the presence of biofilm were analyzed using a Bayesian estimation approach as implemented in the brms R package.^44,45 Convergence and model fit were evaluated using recommendations by Gelman et al.⁴³ Expected log pointwise predictive density was used to compare model fit. The Student distribution was best fit for all continuous dependent variables, whereas a Bernoulli distribution was best fit for presence of biofilm owing to its dichotomous nature.

A Bayesian approach, with its focus on identifying plausible models and parameters, was chosen based on several advantages. For example (1) more can be learned about parameter estimates and model fit; (2) better small sample performance and large sample theory is not needed; (3) the approach focuses on the direct quantification of uncertainty, rather than testing specific null hypotheses given an expected (null hypothesis) distribution; and (4) Bayes estimation uses a full information approach, which takes advantage of all information available and is robust to missing data under the less stringent missing at random (rather than MCAR) assumption. The strength of association calculated by Bayes factor (BF) was interpreted based on Jeffreys⁴⁶ and Makowski et al.⁴⁷

RESULTS

Participants' characteristics

The mean age was 71.7 (SD = 9.6), 64 (55%) were men, and 94 (80%) were white. Participants had VLUs for an average of 321 days (SD = 499), the mean CCI was 5.74 (SD = 1.96), and the mean BMI was 34.1 (SD = 11.9). Participants' characteristics are described in Table 1.

Table 1.

Participant characteristics (N = 117)

Variable	Mean (SD) or Frequency (%)	Range
Age	71.7 (9.6)	55–92
Wound duration, days	321 (499)	20–2,944
BMI	34.1 (11.9)	15.4–84.1
CCI	5.74 (1.96)	2–11
Antibiotic use
No	46 (39)
Yes	71 (61)
Pain medication use
No	37 (32)
Yes	80 (68)
Sex
Male	64 (55)
Female	53 (45)
Marital status^a
Married	59 (50)
Divorced	25 (21)
Widowed	18 (15)
Never married	14 (12)
Unmarried couple	1 (1)
Race
White	94 (80)
African-American	23 (20)
Hispanic
No	114 (97)
Yes	3 (3)
Smoking history
Never smoked	58 (50)
Used to smoke	49 (42)
Current smoker	10 (8)
Education
Elementary school	3 (3)
Some high school	10 (8)
High school graduate/GED	29 (25)
Some college or technical school	37 (31)
College graduate	36 (31)
Prefer not to answer	2 (2)
Income^a
< $25,000	31 (26)
$25,000 to $44,999	17 (14)
$45,000 to $64,999	11 (9)
$65,000 to $84,999	10 (8)
$85,000 to $94,999	5 (4)
> $94,999	9 (8)
Prefer not to answer	34 (29)
Week healed^a
2	7 (6)
4	9 (8)
6	15 (13)
8	16 (14)
Not healed	70 (60)

Does not sum to 100% because of rounding.

BMI, body mass index; CCI, Charlson comorbidity index; SD, standard deviation.

Characteristics of wound-related factors and symptoms over time

The wound area, presence of biofilm, total bacteria, level of serum CRP, and wound-related symptoms (pain, fatigue, exudate, itching, and edema or swelling) over time are described in Table 2. Patients with nonhealed wounds experienced more fatigue, pain, exudate, itching, and edema or swelling and larger wound areas over time. Serum CRP was relatively higher in participants with nonhealed than in participants with healed wounds over time (8.79–4.79, 11.15–10.30 mg/L, healed, nonhealed with no biofilm, respectively), whereas the number of total bacteria was fairly constant over time in both healed and nonhealed wounds.

Table 2.

Characteristics of wound-related factors and symptoms (healed group vs. nonhealed group)

Variable	Healed Group		Nonhealed Group
Variable	No	Yes	No	Yes
Presence of biofilm
Baseline	12 (67%)	6 (33%)	24 (59%)	17 (41%)
Week 2	11 (65%)	6 (35%)	21 (51%)	20 (49%)
Week 4	6 (50%)	6 (50%)	18 (58%)	13 (42%)
Week 6	4 (67%) NA	2 (33%)	13 (50%)	13 (50%)
Week 8		NA	17 (65%)	9 (35%)

Variable	Mean (SD)	Range	Mean (SD)	Range
Wound area, mm
Baseline	917 (3,182)	3.1–21,750	3,225 (7,091)	23.6–48,950
Week 2	782 (3,150)	0–21,726	2,621 (6,124)	54–47,600
Week 4	646 (2,951)	0–18,750	2,529 (5,236)	42–35,376
Week 6	108 (220)	0–900	2,211 (5,200)	4–36,450
Week 8	0 (0)	0–0	2,033 (3,924)	6–18,400
Log₁₀ total bacteria
Baseline	3.22 (2.12)	0–7.24	4.22 (2.00)	0–7.69
Week 2	3.05 (2.19)	0–6.70	4.23 (2.08)	0–7.82
Week 4	3.30 (2.30)	0–6.56	4.34 (2.30)	0–8.12
Week 6	4.95 (1.50)	2.59–6.72	4.41 (2.21)	0–7.85
Week 8	NA	NA	3.80 (2.18)	0–7.68
Serum CRP, mg/L
Baseline	8.79 (23.12)	0.20–157	11.15 (17.74)	0.38–136.0
Week 4	5.68 (5.83)	0.49–26.3	11.41 (14.57)	0.42–87.2
Week 8	4.97 (3.08)	3.17–10.9	10.30 (20.21)	0.35–143.9
Fatigue^a
Baseline	2.25 (2.22)	0–8.33	3.07 (2.56)	0–9.22
Week 2	2.32 (2.26)	0–8.11	3.09 (2.59)	0–10
Week 4	1.88 (2.17)	0–9.44	3.09 (2.60)	0–10
Week 6	2.08 (2.60)	0–9.22	2.97 (2.56)	0–10
Week 8	2.39 (3.23)	0–9.00	2.94 (2.74)	0–10
Pain score^b
Baseline	6.60 (6.10)	0–20	6.71 (5.57)	0–19
Week 2	5.89 (6.05)	0–20	7.06 (5.21)	0–17
Week 4	4.50 (4.83)	0–17	5.92 (5.10)	0–20
Week 6	4.09 (4.44)	0–12	5.39 (4.94)	0–20
Week 8	3.8 (4.61)	0–11	4.64 (4.98)	0–16
Exudate^b
Baseline	2.96 (2.57)	0–10	3.41 (2.43)	0–10
Week 2	2.09 (1.92)	0–7	3.08 (2.36)	0–10
Week 4	1.67 (2.03)	0–10	3.03 (2.52)	0–9
Week 6	1.00 (1.14)	0–4	2.58 (1.98)	0–8
Week 8	1.9 (3.03)	0–10	2.37 (2.29)	0–9
Itching^b
Baseline	1.81 (2.92)	0–10	2.53 (3.16)	0–10
Week 2	2.00 (2.43)	0–9	2.90 (3.14)	0–10
Week 4	1.47 (2.12)	0–10	2.64 (2.85)	0–10
Week 6	1.48 (2.06)	0–8	1.90 (2.12)	0–8
Week 8	1.30 (2.11)	0–7	2.02 (2.33)	0–8
Edema^b
Baseline	2.85 (3.06)	0–10	2.96 (2.69)	0–10
Week 2	2.17 (2.63)	0–10	2.36 (2.42)	0–10
Week 4	1.53 (2.10)	0–10	2.47 (2.47)	0–9
Week 6	1.67 (2.01)	0–7	2.20 (2.31)	0–9
Week 8	0.8 (1.47)	0–4	1.84 (2.18)	0–10

Brief Fatigue Inventory: 9 items with 0- to 10-point scale.

Pain score, Exudate, Itching, and Edema from Toronto Symptom Assessment System having 9 dimensions with 0- to 10-point scale.

CRP, C-reactive protein.

Associations with presence of biofilm for serum CRP and wound symptoms

There was modest (BF = 2.99, 95% CI [−0.15 to 0.98]) evidence for healed wounds having lower odds for the presence of biofilms over time during the study period than nonhealed wounds, after controlling for antibiotic use (Tables 2 and 3). There was strong evidence for a positive association between total bacteria count and the presence of biofilms (BF >1,000, controlling for antibiotic use). There was also moderate evidence for higher mean serum CRP in participants with biofilm present (BF = 4.3). After controlling for pain medication use, there was moderate evidence that the pain score was higher in the presence of biofilm (BF = 5.12). In addition, there was moderate evidence that exudate was higher in the presence of biofilm (BF = 8.49). However, there was no support for an association between presence of biofilm and fatigue, after controlling for CCI and age or for an association between the presence of biofilm and itching and edema (see details in Table 3).

Table 3.

Results of Bayes mixed models

Response Variable	Effect	Estimate ^a (95% CrI)	PD, %	BF ^b
Biofilm presence	Healed^c	−0.456 (−1.85 to 0.83)	75	2.99
Biofilm presence	Antibiotic use^d	0.064 (−1.24 to 1.36)	54	1.15
Wound area^e	Biofilm presence^f	0.002 (−0.08 to 0.08)	52	1.07
Total bacteria^e	Biofilm presence^f	1.41 (0.87 to 1.94)	100	>1,000
Total bacteria^e	Antibiotic use^d	−0.021 (−0.75 to 0.75)	52	1.09
Serum CRP^e	Biofilm presence^f	0.067 (−0.09 to 0.22)	81	4.3
Pain	Biofilm presence^f	0.606 (−0.63 to 1.82)	84	5.12
Pain	Pain Med use^g	3.73 (1.75 to 5.75)	100	>1,000
Fatigue	Biofilm presence^f	−0.025 (−0.49 to 0.45)	54	1.18
	CCI	0.451 (0.12 to 0.79)	100	249
	Age	−0.106 (−0.17 to −0.04)	100	999
Exudate	Biofilm presence^f	0.358 (−0.20 to 0.92)	89	8.49
Itching	Biofilm presence^f	0.054 (−0.46 to 0.55)	59	1.45
Edema	Biofilm presence^f	−0.090 (−0.62 to 0.45)	63	1.72
	CCI	0.356 (0.07 to 0.66)	100	>1,000
	Age	−0.010 (−0.16 to −0.04)	99	109

Linear model regression weight. Presented estimates were after controlled other variables.

Bayes factor: >100 = extreme evidence for H1; 30–100 = very strong evidence for H1; 10–30 = strong evidence for H1; 3–10 = moderate evidence for H1; 1–3 = anecdotal evidence for H1; 1 = no evidence.

Effect for healed wound group compared with nonhealed wound group.

Effect for not using antibiotic relative to antibiotic use.

Log₁₀ transformed values.

Effect for biofilm positive relative to biofilm negative.

Effect for pain medication use relative to not using pain medications.

95% CrI, 95% credibility interval; PD, probability of direction.

Other associations with pain, fatigue, itching, or edema

There was very strong (BF >1,000, 95% CI: [−0.22 to 0.07]) evidence for pain scores to be higher in the group using pain medication. Of note, there was extremely strong evidence for patients with higher CCI scores reporting higher fatigue (0.451, 95% CrI [0.12 to 0.04], BF = 249) after controlling for biofilm presence and age, and older patients reporting higher fatigue (−0.106, 95% CrI [−0.17 to 0.04], BF = 999) after controlling for biofilm presence and CCI. There was strong evidence for patients with higher CCI scores to evidence greater edema (0.356, 95% CrI [0.07 to 0.66], BF >1,000) after controlling for biofilm presence and age, as well as in younger patients (−0.010, 95% CrI [−0.16 to 0.04], BF = 109) after controlling for biofilm presence and CCI (see details in Table 3).

DISCUSSION

We described the associations between systemic inflammation, wound-related symptoms, wound-related factors, including biofilm, and wound healing over the 8-week time frame of the study. As far as we know, this is the first study addressing these associations in human subjects with CVLU over time.

The association between biofilm and wound healing

Patients with wounds that healed during the 8-week study period had lower odds for biofilm presence than patients with nonhealing wounds. In other words, the presence of biofilm may interfere with wound healing. However, presence of biofilm was not associated with wound area, which indicates that the longer time to heal may be a consequence of the biofilm itself rather than wound areas.

In terms of wound area, an association between presence of biofilm and wound area was not supported. Considering the study participants received weekly wound debridement, which may have inhibited the formation of biofilm, this could have affected the association between biofilm presence and wound healing in this study. Another factor that could have influenced wound healing was disrupted inflammatory response owing to the individual's other medical conditions (e.g., comorbidities) or other factors (age and obesity, etc.). Therefore, facilitating an appropriate inflammatory response (e.g., controlling hyperglycemia in patients with diabetes mellitus) combined with weekly debridement may promote wound healing.

Association between biofilm and symptoms

The presence of biofilm was associated with wound-related symptoms such as pain and exudate over time. Weekly sharp debridement that effectively controls biofilm formation may reduce the levels of pain and exudate. Up-to-date recommendations for chronic wound treatment have addressed pain management in detail from the comfort perspective.⁴⁸ Wound-related symptoms such as fatigue, pain, exudate, itching, and edema or swelling have been described as factors related to the quality of life in the VLU population.^49,50 There is a lack of research linking biofilm or wound molecular environment with wound-related symptoms. When patients experience persistent wound-related symptoms, such as pain, wound exudate, and itching, clinicians should consider the presence of biofilm. However, because we did not compare wound symptoms between patients with biofilm and those without, further study is needed to validate our suggestion.

Symptoms decreased over time in the healed group (Table 2), but pain scores were higher in the biofilm present group (Table 3). Therefore, if a wound is healing, its symptoms, such as pain and exudate, should be decreasing, according to the study findings. However, if symptoms are consistent or increasing, clinicians should consider the possibility that wound healing is not progressing, reassess the patient's condition, and revise the individual care plan depending on patient's condition (e.g., treat an infection or reduce inflammation by controlling hyperglycemia) to promote wound healing.

Association between biofilm and systemic CRP

Serum CRP had positive associations with the presence of biofilm, that is, individuals with biofilm in their wounds tended to have higher serum CRP levels. It is believed that biofilm can delay wound healing by impairing reepithelialization,^17

–20 skin barrier function,²⁴ and modulating inflammatory processes.⁵¹ This is further complicated by complex interactions with patient-specific conditions such as aging, stress, and comorbidities.¹⁰ In this study, wounds that healed during the 8-week study period had lower odds for biofilm presence than wounds that did not heal. However, the absence of biofilm did not assure healing, nor did the presence of biofilm prevent healing. Perhaps, when biofilm is consistently debrided, appropriate inflammatory responses are allowed to develop, and wound healing trajectories continue toward healing instead of becoming chronic. Therefore, the study results suggest that serum CRP levels may indicate appropriate inflammatory processes in chronic wounds treated with weekly sharp debridement.

Current evidence has not fully described the associations among serum CRP, wound biofilm, and wound healing over time. In contrast with our results, a study measured serum inflammatory markers to assess the influence of P. aeruginosa on healing rate among CVLU indicated that neither serum CRP levels nor the presence of P. aeruginosa was associated with wound healing.⁵² However, comorbidities, likely influence the level of serum CRP. Further research is needed to investigate the associations between serum CRP, comorbidities, biofilms, and wound healing.

Biofilm, total bacteria, wound-related symptoms, and other factors

This study demonstrated that biofilm was strongly associated with the number of total bacteria. Presence of biofilm was also found to be associated with wound-related symptoms such as fatigue and edema or swelling. The study revealed that fatigue and wound edema are more severe in individuals with more comorbidities and younger age. However, there is currently no corroborating evidence to support these findings in patients with CVLU. Thus, further research is required to verify these results.

An association between antibiotic use and biofilm presence was not supported (BF = 1.15). That could be a result of clinical judgment based on wound characteristics, with wounds that had signs of infection or nonhealing being more likely to receive systemic antibiotics. In addition, wound characteristics, especially progress toward healing, may have led to the clinical decision that antibiotics were not needed. Although there is inconsistent evidence for the benefit of using antibiotics on chronic wounds, clinicians commonly prescribe antibiotics based on wound appearance and the result of wound culture.^6,53 Recently, some researchers have suggested combining antibiotics with wound debridement and treatment of the underlying comorbidities to treat wound infections.^54,55 Hence, when prescribing antibiotics for wounds, clinicians should carefully consider benefits of antibiotic treatment and only administer them when necessary.

Limitations

The study's participants had varied wound duration before entry into the study and had multiple comorbidities. Owing to attrition of study participants as wounds healed, sample sizes available to estimate strength of associations decreased over time, with the remaining sample increasingly composed of wounds that were resistant to healing. The findings have limited generalizability because the participants were recruited from the one regional wound clinic in the Southeast and experienced the same treatment protocol.

SUMMARY

There was support for an association between the presence of biofilm and wound-related symptoms and systemic CRP in patients with CVLU. Clinicians should consider the association of biofilm presence with systemic inflammation (serum CRP) and wound-related symptoms during treatment to decide on the most appropriate treatment modality for the patient. We present wound care strategies based on the study results and modified from Raffetto et al. (Fig. 1).⁵⁶ Careful observation of the trends of serum CRP and wound-related symptoms during the treatment may provide feedback regarding the effectiveness of the clinician's treatment plan for patients with CVLU.

Figure 1.

Summary of wound care strategy for chronic venous leg ulcers. Modified from Raffetto et al.⁵⁷

INNOVATION

Understanding the association between biofilm, wound-related symptoms and inflammatory markers may give clinicians a clue to predict wound healing trajectory. The study results indicated that (1) biofilm may not have much of an influence under weekly sharp debridement. (2) Trends of the symptom severity and level of systemic CRP may be biobehavioral markers for predicting wound healing trajectories during the weekly sharp debridement. (3) More research is needed to explore the factors in addition to biofilm associated wound healing under weekly sharp debridement.

KEY FINDINGS

Biofilm may delay wound healing, but maintaining proper inflammatory pathways may promote the healing of wounds with biofilm.

Biofilm may not have much of an influence on wound healing under weekly sharp debridement.

Symptom severity and level of systemic CRP may be biobehavioral markers for predicting wound healing trajectories during the weekly sharp debridement.

Longitudinal observation of the behavior of serum CRP and wound-related symptoms may help clinicians predict wound healing trajectories and choose treatment modality.

More research is needed to explore the factors in addition to biofilm associated wound healing trajectories under weekly sharp debridement.

ACKNOWLEDGMENTS AND FUNDING SOURCES

None declared. This study was supported by the National Institutes of Health (NIH), National Institute of Nursing Research (NINR), Grant No. R01:NR016986. Also, Junglyun Kim's effort to this work was supported by the National Research Foundation of Korea, Grant No. NRF-2022R1C1C1006659.

AUTHOR DISCLOSURE AND GHOSTWRITING

All authors declared no conflict of interest. All authors wrote, critically reviewed, and revised the article. No ghostwriters were used to write this article.

ABOUT THE AUTHORS

Junglyun Kim, PhD is an Assistant Professor at the Chungnam National University College of Nursing in the South Korea and a Courtesy Assistant Professor at the University of Florida College of Nursing. Dr. Kim has extensive clinical experience in chronic wounds and knowledge in biobehavioral symptom science. Joyce Stechmiller, PhD is a Professor at the University of Florida College of Nursing. Dr. Stechmiller has extensive experience and expertise in wound science, with special emphasis on biobehavioral approaches to enhancing wound healing. Michael Weaver, PhD is Professor and Associate Dean for Research at the University of Florida College of Nursing. Garth James, PhD is an Associate Research Professor, Medical Biofilms Laboratory Center for Biofilm Engineering at Montana State University. Phill Stewart, PhD is Regents Professor, Chemical and Biological Engineering at Montana State University. Debra Lyon, PhD is a Professor and the Kirbo Endowed Chair and Executive Associate Dean of Biobehavioral Nursing Science of the University of Florida College of Nursing. Dr. Lyon's research focus on symptom management, including complementary modalities, in women with breast cancer and chronic illnesses. She is an expert in biobehavioral research, including molecular markers of inflammation and genetic markers.

Footnotes

Abbreviations and Acronyms

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