Abstract: Topical application of a combination of nitrofurazone and rifamycin in contaminated wounds is widely used in Turkey with considerable success. Although nitrofurazone is generally abandoned in European countries and the United States, rifamycin is used for topical wound care in many European countries. However, the experimental data is lacking. In this study, full-thickness circular dorsal wounds were created in rats. Three experimental and 2 control groups were constructed. After creation of the defects, the wounds were treated with topical nitrofurazone in the first experimental group, topical rifamycin in the second experimental group, and a combination of topical nitrofurazone and rifamycin in the third experimental group. In the first control group, the wounds were treated with a combination of bacitracin and neomycin, and in the second control group, the wounds were treated with daily cleansing using 0.9% NaCl. The progression of wound healing was measured and documented using photography and millimetric scales. The results demonstrated that the nitrofurazone-treated wound group had significantly delayed full-thickness wound healing. No significant differences were observed among the other groups. The interesting finding is that the combination of nitrofurazone and rifamycin abolished the delayed healing effect observed in the nitrofurazone-treated group.

       Topical antibiotics have been used in wound care for many years. A number of reports have indicated the effectiveness of topical antibiotics for prevention of infection in surgical and traumatic wounds.^1–3 However, many authors do not prefer the use of topical antibiotics because of the risk of development of resistance and possibility of hypersensitivity reactions.^1,4 Nitrofurazone- and rifamycin-absorbed gauze dressing is commonly used in Turkey, but the literature and data supporting this mode of therapy is lacking. Although some reports in the literature discuss the topical use of rifamycin in wounds,⁵ sufficient data is not available. The authors aimed to determine the effects of topical use of these agents in a full-thickness wound model in rats.

Materials and Methods

       Fifty Wistar rats weighing 250–350 g were used in the study. The animals were kept in standard metal cages and provided with food and water ad libitum. Anesthesia was administered intramuscularly with xylazine hydrochloride (Rompun 2%, Bayer Kimya San. Ltd. Sti., Istanbul, Turkey) and with 5 mL/kg ketamine hydrochloride (Ketalar, EWL Eczacibasi Warner Lambert Ilaç Sanayi ve Ticaret A.S., Istanbul, Turkey). After cutting the hairs, 2 circular full-thickness defects with a diameter of 1.5 cm were created on the dorsal left superior and right inferior regions of the rats (Figure 1).

Figure 1

In all groups, daily wound care was performed by cleansing the wound with 0.9% NaCl solution. The surface area of each circular defect was 176.6 mm². The dimension of the gauze used in wound care was 30 cm x 34 cm. For each rat, 1/250 of the gauze was used making the surface of the gauze approximately 4 cm². The experimental and control groups contained 10 rats (n = 10). In this study, the authors utilized a clinical method that has been in use for more than 10 years and is a traditional technique used in Turkey. For this reason, a secondary occlusive dressing was not used to obtain a moist environment.
       Experimental groups. Nitrofurazone group. To mimic clinical use, a 56-g tube of nitrofurazone (Furacin, 0.2%, Eczacibasi Ilaç Sanayi ve Ticaret A.S., Istanbul, Turkey) was simply absorbed by 2 gauzes in the nitrofurazone group. The average nitrofurazone dosage was approximately 0.1 g for each wound.
       Rifamycin group. The standard gauze was soaked with 1 ampule of rifamycin (Rif 250 mg injectable form, Koçak Ilaç A.S., Istanbul, Turkey). The average dosage of rifamycin was approximately 1 mg for each wound.
       Nitrofurazon-rifamycin combination group. The above methods were combined in this group.
       Control groups. Neomycin-bacitracin group. Animals in the neomycin-bacitracin group received 1/256 of the standard gauze that had absorbed a combination of neomycin and bacitracin (Thiocilline pom 10 g, Abdi Ibrahim Ilaç Sanayi, Istanbul, Turkey). The average dosage was 0.08 g per wound. The neomycin-bacitracin group was selected as a control group because this formula is widely accepted for wound care in Europe and the United States.
       No treatment group. Wounds in this group were merely cleansed daily with isotonic NaCl.
       All wounds were cleansed with NaCl jet lavage prior to treatment with agents.
       Evaluation of the wounds. The course of wound healing was assessed by drawing the circumference of the wound on acetate paper with permanent 0.3-mm markers (Figure 2). Measurements were performed on Days 3, 6, 9, 12, and 30. The wounds were evaluated every 3 days with photography and millimetric measurement. However, during daily treatments, when complete healing was observed, the wound was recorded as completely healed. Statistical analysis was performed only on data from the “exact” day of healing. Evaluations were made for the purpose of obtaining information about the course of healing.

Figure 2

       Calculation of the wound surfaces was performed with a special software (Surfer 7.0, Golden Software, Golden, Colo) developed for map drawing in mining and geology. The drawings obtained from the wounds were transferred to the computer.
       Statistical evaluation. The data derived from the study was transferred to Statistical Package for the Social Sciences (SPSS) software (SPSS Inc., Chicago, Ill). In order to eliminate healing differences between the upper and lower wounds, wounds were also analyzed separately. The Kruskal-Wallis test was performed to compare groups. The significant values obtained from this test were assessed with the Mann-Whitney U test.

Results

Results for each group were as follows:
• Nitrofurazone group. The earliest wound healing was observed on Day 18, the latest was observed on Day 28, and the average wound closure time was 22.1 days.
• Rifamycin group. The earliest wound healing was observed on Day 15, the latest was observed on Day 24, and the average wound closure time was 19.6 days.
• Nitrofurazone-rifamycin combination group. The earliest wound healing was observed on Day 14, the latest was observed on Day 22, and the average wound closure time was 17.5 days.
• Neomycin-bacitracin combination group. The earliest wound healing was observed on Day 15, the latest was observed on Day 22, and the average wound closure time was 18.1 days.
• No treatment group. The earliest wound healing was observed on Day 17, the latest was observed on Day 24, and the average wound closure time was 19.4 days.
       The mean healing times and the standard deviations are shown in Table 1.

Table 1

       Statistical relationships among the groups. Upper and lower wounds were evaluated separately. In both upper and lower wound groups, the groups were compared with the Kruskal-Wallis test. The differences among the groups were significant (P = 0.01 for the upper wound group; P = 0.007 for lower wound group).
       Upper wounds. Five groups were compared with the Mann-Whitney U test:
• Between the nitrofurazone group and the rifamycin group (P = 0.109)
• Between the nitrofurazone group and the nitrofurazone-rifamycin combination group (P = 0.009)
• Between the nitrofurazone group and the neomycin-bacitracin combination group (P = 0.003)
• Between the nitrofurazone group and the no treatment group (P = 0.04).
The other group comparisons were found to be insignificant (P > 0.05).
       Lower wounds. Five groups were compared with the Mann-Whitney U test:
• Between the nitrofurazone group and the rifamycin group (P = 0.079)
• Between the nitrofurazone group and the nitrofurazone-rifamycin combination group (P = 0.009)
• Between the nitrofurazone group and neomycin-bacitracin combination group (P = 0.005)
• Between the nitrofurazone group and the no treatment group group (P = 0.016).
       The other group comparisons were found to be insignificant (P > 0.05).
       Interestingly, no statistical significance appeared between the nitrofurazone group and the rifamycin group in both upper and lower wound groups. Although the nitrofurazone group was significantly different from the other 3 groups, the nitrofurazone and rifamycin groups were not significantly different from each other, and the rifamycin group was not significantly different from the other 3 groups. This seems to be a bias. However, the authors attribute this to the low number of wounds. The other statistical values seemed to be rational, and the statistical results of the upper and lower wounds were comparable. Figures 3 and 4 show the error bars of both groups.
       The wounds in the nitrofurazone-treated group healed slower than the other groups and this result was statistically significant. No statistically significant results were encountered among the other groups. While the wounds treated with nitrofurazone alone healed slowly, wounds treated with nitrofurazone-rifamycin combination healed as fast as the other groups.

Figures 3–4

Discussion

       There is no data in the literature on the use of the combination of nitrofurazone and rifamycin in wound care. The only report, written by the authors of the present study, demonstrated the in-vitro efficacy of a combination of nitrofurazone and rifamycin.⁶ Several recent reports showed the negative influence of nitrofurazone on wound healing.^7–9 However, some contradiction about the effect of nitrofurazone ointment on wound healing exists. In some studies, nitrofurazone ointment was utilized as a standard agent for comparison of different agents.¹⁰ The authors could not locate an experimental study on the effect of rifamycin on wound healing. The only report found in the literature is the abstract of a French manuscript. In that particular study, the effect of topical rifamycin on 268 patients with hand injuries requiring surgery was evaluated. The result favored the use of rifamycin over povidone iodine, and increased healing was observed with topical rifamycin.⁵
       In the present study, the authors found that although isolated use of nitrofurazone caused delay in wound healing in the full-thickness wound model, combined use of nitrofurazone with rifamycin did not cause a delay in wound healing. Rifamycin eliminated the retardation effect of nitrofurazone. This is an interesting finding that requires further research. Rifamycin may have chemically blocked some of the nitrofurazone features that caused a delay in healing.
       Nitrofurazone is nearly abandoned in European countries and in the United States because of its hypersensitivity reactions. However, in a recent study, it was shown that the rate of hypersensivity reactions was 2% in patients with surgical and traumatic wounds.¹¹
       Nitrofurazone is an inexpensive broad-spectrum antibacterial agent. A disadvantage of nitrofurazone is the resistance of Pseudomonas aeruginosa to this drug. However, when it is combined with rifamycin, P aeruginosa apparently becomes susceptible to this combination.⁶

Conclusion

       When nitrofurazone and rifamycin are combined, many advantages become apparent. The antibacterial spectrum increases, the risk of drug resistance development decreases, and, based on the present study, the risk of wound healing delay decreases.