Dilution of Open Fracture Grade IIIA of the Lower Leg Using Normal Saline 0.9% Mixed with Honey Compared With Normal Saline 0.9% to the Bacterial Growth

The incidence of traffic accidents in Indonesia has increased from 2007 (48.508 cases) to 2008 (59.164 cases) and in 2009 (62.960 cases). One of many results of these traffic accidents is fracture of the long bone [6]. These fractures are at risk for an impaired healing due to any connection with external environment. Contamination incidence of open fracture wounds in Dr. Hasan Sadikin hospital in 1994 is 55.1% and increased 91% in 1998.


Introduction
The incidence of traffic accidents in Indonesia has increased from 2007 (48.508 cases) to 2008 (59.164 cases) and in 2009 (62.960 cases). One of many results of these traffic accidents is fracture of the long bone [6]. These fractures are at risk for an impaired healing due to any connection with external environment.
Contamination incidence of open fracture wounds in Dr. Hasan Sadikin hospital in 1994 is 55.1% and increased 91% in 1998.
One of the important management of handling patients with open fractures is cleansing the wound, besides administration of appropriate antibiotic according to the bacterial culture, and immobilizing fracture segments with external fixation [7]. It is one of the keys to prevent any infection in open fractures because it can dilutes the concentration of bacteria, removing foreign bodies in the wound and reduce the number of bacterial colonies.
Various ways have been attempted to reduce the number of colonies of germs in patients with lower extremity wounds open fractures grade III A, by cleansing the wound with 0.9% NaCl, washing wounds using boiled water, using a mixture with povidone iodine, but the infection rate is still high in patients with open fractures of lower leg grade III A, therefore the author have an idea of using a mixture of 0.9% NaCl and honey [8].
The purpose of cleansing the wound with 0.9% NaCl is to dilute the concentration of bacteria, remove foreign bodies in the wound, so that when debridement is done, the number of colonies of bacteria and germs has been reduced [9]. Honey has an antibacterial activity and inhibine factors which can decrease the incidence of infection so that honey can be used in patients with open fractures grade IIIA. Hydrogen peroxide is produced by honey after reaction with the enzyme glucose oxidase which becomes active when the honey is diluted. The use of honey as a mixture with NaCl 0.9% in irrigating fracture lower leg injuries grade IIIA will help lower the number of colonies of germs that will reduce the incidence of infection. The purpose of this study was to evaluate the use of a mixture of NaCl 0.9% -honey in wound irrigation in open fractures of lower leg grade IIIA in the ER, whether it is the same, better or worse in reducing the number of bacteria colonies compared to using 0.9% NaCl [10].

Method
This research is an experimental study by comparing the wound diluted using 0.9% NaCl and honey mixture and 0.9% NaCl alone. These research subjects have to meet the inclusion and exclusion criteria. Inclusion criteria included: age 16-55 years, patients injured lower limb fractures open grade IIIA caused by traffic accidents who have not received medical procedures such as irrigation and administration of prophylactic antibiotics since the accident, which is less than or equal to 6 hours [11]. The exclusion criteria is when the subject is under treatment of antibiotics or corticosteroids.
The procedure of the study include informed consent and filling the consent form, identity record, wound irrigation by using a mixture of 0.9% NaCl and honey for the first group and irrigation of the wound with NaCl 0.9% only for the second group, and last, sample taking with simple random sampling.
Wound cleansing on the subjects of the first group use a mixture of 0.9% NaCl and honey, prepared by injecting 100 grams of honey (weighed with digital scales) by using a 50cc sterile syringe container into 500mL kolf of NaCl 0.9% at Hasan Sadikin Hospital emergency department. Wound cleansing on the subjects in the second group use plain 0.9% NaCl. Irrigation technique is based on pressure irrigation system for open fracture wounds that uses as much as 9 liters of fluid conducted in the ER by pressing the packaging of 0. 9% NaCl until it was emptied [12].
A sample was taken from the base of the wound before and after dilution by using a sterile cotton swab stick at the bottom of the wound from both groups of research subjects and was put into a sterile test tube by author assisted by a trained assistant then taken to the laboratory of Clinical Pathology Department of Clinical Microbiology Division Hasan Sadikin General Hospital.
The sample was taken from the test tube by using a loop stick ose of 10 microns, then the sample is grown / cultured in a petri dishes with Mueller-Hinton media and kept in an incubator at 37 °C for 24 hours [13]. After 24 hours, the growing bacteria is counted.
The bacteria count is carried on the 2 nd day directly by counting the number of colonies one by one based on the growing bacteria on the petri dishes with Mueller-Hinton media [14]. Furthermore, the data acquired is analyzed with non-parametric statistical tests Wilcoxon signed-ranks for the paired data and Mann-Whitney test for independent data in setting of p-value by using SPSS version 13.0 [15].

Result
Analyzing the obtained statistical characteristics such as age, gender, and extensive wounds is in accordance with (Table 1). Based on (Table 2), note a smaller number of bacteria colonies after dilution with NaCl 0.9% and honey compared with before dilution (negative difference) is found in 12 samples and has the rank number (R1) of 78.00, while 2 samples showing the same number of bacteria colonies after and before dilution, and no samples shows a greater number of bacteria colonies after dilution (positive difference). Statistical two paired sample tests were performed with SPSS, results in Z value of -3.163. The difference of two samples tested by Asymp Sig (2-tailed) obtained the value 0.002. This means that there is significant difference between the data before and after the group was given the dilution of NaCl 0.9% and honey mixture ( Table 3).
The same experiment was done using 0.9% NaCl without the mixture of honey. A smaller number of bacteria colonies after dilution with NaCl 0.9% compared than before dilution (negative difference) is found in 13 samples and has the rank number (R1) of 95.00, no samples showing the same number of bacteria colonies after and before dilution, and a greater number of bacteria colonies after dilution (positive difference) is found in 1 sample and has the rank number (R2) of 10.00 (Table 4). Statistical two paired sample tests were performed with SPSS, results in Z value of -2.765. The difference of two samples tested by Asymp Sig (2-tailed) obtained the value 0.006. This means that there is significant difference between the data before and after the group was given NaCl 0.9% dilution (Table 5). U Mann-Whitney test was done with the help of SPSS version 13.0 app, the result is as following ( Table 6).    There is a significant difference in the reduction in the number of colonies of bacteria in the samples that were given 0.9% NaCl and honey, as compared to the samples that were given 0.9% NaCl (Table 7).There was no significant difference between pretest data group classes that were given a mixture of NaCl 0.9% -honey and pretest data that were given NaCl 0.9% alone. But there are significant differences between post test data group classes by a mixture of 0.9% NaCl with honey-class and post-test data by NaCl 0.9%.

Discussion
The purpose of open fractures management is to restore the normal or optimal function of a limb as soon as possible. Thus, it is important to prevent the wound from infection. One of the attempts is cleansing the wound to reduce the number of bacteria colonies so it can dilute the concentration of contaminants and bacteria.
The nature of honey with a high level of osmolarity is caused by the high sugar levels which dilute the product from microbial and stop the growth of all microbial species. It contains glucose oxidase enzyme so that when the honey is diluted, it will produce a low concentration hydrogen peroxide which has an antiseptic and antimicrobial effect. Honey can also create a moist environment by reducing the lymph fluid from the wound tissue through a high osmotic work. Debridement effect of honey is caused by protease enzyme by releasing hydrogen peroxide. In this study, there are 28 patients, 14 patients underwent dilution with a mixture of 0.9% NaCl and honey, and 14 other patients underwent dilution with 0.9% NaCl alone. The data were compared and it showed that dilution with a mixture of 0.9% NaCl with honey will reduce the number of bacteria colonies significantly compared to those wounds diluted with NaCl 0.9% alone.