Is Vancomycine Still a Choice for Chronic Osteomyelitis Empirical Therapy in Iran?

Background Pyogenic bacteria and especially Staphylococcus aurous (S. aurous) are the most common cause of chronic osteomyelitis. Not only treatment protocol of chronic osteomyelitis occasionally is amiss but also this malady responds to treatment difficultly. Objectives This study investigates antibiotic resistance pattern of S. aurous isolated from Iranian patients who suffer from chronic osteomyelitis by two methods: disk diffusion (Kirby bauyer) and E-test (Epsilometer test) to find Vancomycin susceptibility and MIC (Minimum inhibitory concentration). Patients and Methods One hundred and thirty one patients who suffer from chronic osteomyelitis which have been referred to both governmental and private hospitals at 2010 were tried out for culturing of osteomyelitis site (sites). Antibiotic susceptibility and MIC of isolated bacteria were investigated by Kirby bauyer and E-test respectively. Results Samples were collected from bone (73.4%), surrounding tissue (14.6%) and wound discharge (12%). S. aureus was isolated from 49.6% of the samples. According to disc diffusion, methicillin resistance S. aureus (MRSA) was 75% and Vancomycin resistance S. aurous (VRSA) was 0% and based on MIC, MRSA was 68.5% and VRSA was 0%. According to MIC experiments, maximum sensitivity was against to Vancomycin (90.2%) and ciprofloxacin (54.4%) respectively but based on disc diffusion, maximum sensitivity was against to Vancomycin (97.7%) and ciprofloxacin (43.2%), respectively (P = 0.001). E-test (9.8%) in comparison with Disc diffusion (2.3%) showed higher percent of intermediate susceptibility to Vancomycin (P = 0.017). Conclusions Comparison of antibiograms and MICs showed that Kirby bauyer technique especially for detection of VISA strains is not reliable comparison with E-test. Already VRSA strains have not detected in Iranian chronic osteomyelitis, Thus Vancomycin is the first choice for chronic osteomyelitis empirical therapy in Iran yet.


Background
osteomyelitis cause to non-responding to short period treatment (1). Presence of collagen leads to microorgan-Osteomyelitis is an invasive and hard-treatable infec ism adhesion to osteon's matrix (2). Various pathogens tious disease conducted to periosteum and cortex of osplay a role in chronic osteomyelitis which depend on teon by aversion canals and it will cause necrosis if peri several factors such as age and medical history. Adults are osteum is infected. Recurrent exacerbations in chronic usually infected by S. aurous and gram negative bacteria (3). There is a variety in the organisms of chronic osteo myelitis in different regions across the globe. In most Eu ropean and North American studies, the most common bone infections have been reported, are Staphylococci and Streptococci with Neisseria gonorrhea, particularly prevalent in North America (4).
More expenses and poor prognosis would be expected when appropriate antibiotic treatment is delayed (5,6). While the responsible organism is not definite, the first choice of antibiotics is empirical. S. aureus, as a main agent of chronic osteomyelitis (7), attaches to osteon's matrix receptors and affects fibronectin, laminin and cialo glycoproteins of matrix (8). Staphylococcus includes 30 species, at least three of which have clinical symptoms as osteomyelitis: S. aurous, S. epidermidis, S. Saprophyticus (7). Considerable controversy still exists over the current and future roles of Vancomycin in controlling serious Methicillin-Resistant Staphylococcus Aurous (MRSA) in fections. Therefore, periodic studies on Vancomycin re sistance S. aurous (VRSA) and Vancomycin-intermediate S. aurous (VISA) are substantial (9). S. aurous strains have reduction teicoplanin susceptibility without a clearly demonstrated reduction in Vancomycin susceptibility (10), while VISA strains have demonstrated reduced tei coplanin susceptibility (11). Nowadays, however, focusing on VISA studies for preventing resistance strains is more valuable compared to VRSA ones. On the other hand, chronic osteomyelitis does have a heavy burden on affected patients and societies. Several orthopedic surgeries and hospitalizations are needed, thus vast expenses of treatment are necessitated in chron ic osteomyelitis sufferer; these factors make low compli ance and consequently low sufferers' consent to antibi otic therapy entirely. Especially in developing countries, excessive antibiotic consuming in these patients yield to S. aurous multidrug resistance strains (12) and generally periodic surveillances on S. aurous as the main etiology of this expensive malady is necessitated to provide appro priate empirical treatment of infections circulating in every geographic region (13). In previous studies epsilom eter test (E-test) comparing with Disc diffusion especially on brain heart infusion agar plate has shown no error in resistant and intermediate susceptibility to Vancomycin for Enterococcus (14), also about another organism such as Haemophilus influenza, E-test is reliable for resistant and intermediate antibiotic susceptibility in comparison with Disc diffusion (15).

Objectives
This study investigates antibiotic resistance pattern of S. aureus isolated from patients who suffered from chronic osteomyelitis by two laboratory methods (Kirby bauyer and E-test).

Subjects
131 patients with osteomyelitis continued for 6 months at least, entered our cross-sectional study. Subjects had been referred to two referral hospitals of Tehran, Iran (one teaching hospital and one private hospital) during the year 2010. None of the subjects had received any anti biotics during 2 last weeks before entering the study. Sub jects who had to receive antibiotics due to their critical conditions, were excluded from the study.

Sampling
All subjects were visited by both infectious disease and orthopedics specialists and site of biopsy was chosen by at least one of them. Bone biopsy, surrounding tissue and wound discharge sampling were done by orthopedic spe cialist at operation room under sterile condition. Sam ples were transferred to laboratory by transport medium straightaway.

Laboratory Analysis
All samples were subcultured onto Mueller Hinton agar plates and incubated at 37°C for 24 hours. A subculture of each strain was suspended in 2 ml of nutrient broth and then adjusted to a turbidity equivalent of 0.

Statistical Analysis
Statistical analyses were done using the chi-square test with statistical package of social sciences, version 16.0 (SPSS Inc, Chicago, IL). A P value of less than 0.05 was con sidered significant.

Results
107 male (81.7%) and 24 female (18.3%) subjects with chronic osteomyelitis were included in this study (male to female ratio was 4.4:1). Mean age was 42.59 years with age range of 10 to 67 years. The most common affected bones in these patients were Tibia (33%) and Femur (27%), fol lowed by Hip (14%), Foot (14%), Humorous (7%) and Malleo lus (5%). The most positive subcultures were obtained from bone (73.4%) and the least ones were taken from wound discharge (12%), while 14.6 percent of positive subcultures were taken from Surrounding tissue. The most common species grown from samples was S. aurous (48.9%). Growth species were categorized in Figure 1. S. aurous had the most sensitivity to Vancomycin (97.7%) and the least sensitivity to Penicillin (7%). Figure 2 summarizes the comparative results of disc diffusion. Antibiotic susceptibility patterns regarding E-test are shown in Figure 3

Discussion
Chronic osteomyelitis has a heavy burden on the affect ed patients and societies. Several orthopedic surgeries and hospitalizations are needed, however, vast expenses of treatment are necessitated in chronic osteomyelitis sufferer (16). In many parts of the world, including Iran, there are not enough studies on the exact scale of caus ative organisms of osteomyelitis. To our knowledge, in Iran as a representative of Middle East, Only two stud ies investigated osteomyelitis characteristics and caus ative organisms (17,18). In the first study, Kalantari et al. analyzed 145 cases of septic arthritis and osteomyelitis in 2007 where 84 cases proved to have osteomyelitis. Age range was 6 days to 15 years with male to female ratio of 1.2:1. Overall, S. aurous was recovered from 61% of posi tive cultures and was the most frequent organism which grew. VISA by disc diffusion (11%) was reported more than our study (2.3%); however, 48% of osteomyelitis cases had arthritis simultaneously. Femur and tibia with 13.7% and 11%, respectively, were the most common bones involved with osteomyelitis, this finding is the same as our study; nevertheless, rate of involvement in these bones was higher in our study. The probable reason is pure osteomy elitis in our survey versus companionship of osteomyeli tis and arthritis in about half of cases in that study. Knee, hip, and ankle consistently account for at least 80 percent of pediatric arthritis (19).
Second study was done on war handicapped chronic osteomyelitis by authors of this paper; in that retrospec tive study, S. aureus was ranked first among causes of os teomyelitis (51.9%) in war handicapped chronic osteomy elitis where all subjects were male (17). Like the present study, the most common involving bones were femur (45%) and tibia (25%) in handicapped chronic osteomy elitis; We have observed almost the same result. S. aurous was the most positive result culture from chronic osteo myelitis (49.6%) among 131 subjects from general popula tion compared to 40 subjects who were war handicapped (51.9%). This similar result can support similar protocol of treatment for war handicapped chronic osteomyelitis and general population patients.
There are several regional studies on S. aurous isolated from several infection sites except osteomyelitis. Mosta favizadeh et al. reported 75% MRSA strains from nosoco mial infections by E-test which is different from our MRSA isolation rate. The probable reason for this can be the difference in location of samples (osteomyelitis versus nosocomial infections) and consequently difference in strains (20); Moreover, this confirms difference in S. aurous strains from different locations so if we are aware of MRSA and VISA and VRSA strains in nosocomial infections of Iran, It is needed to carry out similar periodical studies on osteomyelitis or other infection sites. Kim et al. performed similar study in Koreans' chronic osteomyelitis. 49 patients with age range of 11 to 79 years and mean age of 47.3 years were examined for efficacy of surgery and antibiotic therapy with a new protocol. The others, however, did not report grown microorganisms details. Male to female ratio was 1.7:1 in their study (21). Demographic data was almost the same as our study but they had a wider age range.
In another study, Orimolade et al. investigated clinico pathological characteristics of Nigerian patients with acute and chronic (76.7%) osteomyelitis. In their study, male to female ratio was 2:1 and age range was above 15 years. S. aureus grew in 80.8% of grown samples (22) Which was more than our finding (49.6%). This higher result might be due to the presence of acute osteomyeli tis among their participants. Eschrichia coli (7.7%), Pseu domonas aero Gina (3.8%) and Klebsiella (3.8%) were the most common germ after S. aurous in Nigerian osteomy elitis, similar to our study, with a difference in ranks; In Iranian osteomyelitis, Pseudomonas aero Gina (11.5%), E. coli (8.4%) and Klebsiella (6.9%) were ranked after S. aureus respectively. In our study the most resistance of S. aurous was to Penicillin (93%) and Amoxicillin (79.5%) and Trimethoprim-sulfamethoxazole (77.3%) based on disc diffusion in Iranian osteomyelitis and the most sensitiv ity to Vancomycin (97.7%) ; Also based on E-test, the most sensitive antibiotic was Vancomycin (90.2%). This pattern underscores the importance of Vancomycin empirical therapy in Iranian osteomyelitis. MRSA was reported 75% by disc diffusion; though significantly, it was determined in a smaller scale by E-test (68.5%). Moreover, disc diffu sion results showed 2.3% strains as VISA which was less than E-test results (9.8%) significantly. These results sup port E-test as a reliable test for osteomyelitis. On the other hand, 5-microgram disk diffusion test is easy to perform but does not give real results about VISA similar to E-test method; certainly, disk diffusion is substantialy enough to determine a clinical choice for antibiotic therapy in chronic osteomyelitis and our findings support that.
Overall, clinical trials of Iranian osteomyelitis are need ed in future to evaluate exact results of E-test by clinical response to antibiotics. Although our study supported Vancomycin empirical therapy, detection of a reliable method for finding a right narrow spectrum antibiotic can induce lower empirical therapy and consequently keep VISA in a low level and prevent construction of VRSA strains in Iranian chronic osteomyelitis.
The attainment of antimicrobial resistance and chang ing patterns of staphylococcal disease have been com mon themes in the staphylococcal literature over the past 50 years and there is not any exception for osteomyelitis causative staphylococci. Therefore, periodic evaluation of VRSA and VISA should be performed in order to be pre pared for VRSA strains and also in case of sudden increas ing in level of VISA strains, by executing more studies on genotype, biological behavior and etc. of new kinds of VISA. There is no report of VRSA in Iranian osteomyelitis; However, according to previous reports of Iranian speci mens, VRSA strains do exist in Iran, (23) although there is no report of VRSA in Iranian osteomyelitis already. So Imipenem and other carbapenems are not suggested es pecially in Iran.
There are no VRSA strains in Iranian osteomyelitis and thus Vancomycin empirical therapy for chronic osteo myelitis is choice for the present time and periodical studies, especially by E-test, can make us aware of VRSA strains' presence and VISA strains'. In case of these two events, serious response should be triggered to alter os teomyelitis treatment protocol.