Santa Rosa Antibiotic Effectiveness Project, Inc.

CURRENT STUDY

Soft tissue infections are a major global health issue. When an inadequate concentration of antibiotic is used to treat a given infection, antibiotic resistant strains of bacteria develop that are increasingly difficult to treat. Patients with MRSA cellulitis of the lower extremity, especially those with impaired circulation, are even more difficult to treat with standard delivery methods of antibiotics because an adequate concentration of antibiotic cannot be delivered to the target tissues.

Previous to this study, Cephalosporin antibiotics were considered to be ineffective in treating MRSA as they had only been tested at the concentrations that were attainable by traditional methods, i.e., oral or intravenous delivery. Our Phase I pilot study has confirmed that Cephalosporin antibiotics are effective against MRSA infections if a high enough concentration can be obtained. Since Cephalosporins are not toxic to local tissues in higher concentrations, a much higher concentration than what is routinely administered before surgery can be given without fear of tissue damage.

This study focuses on a technique wherein Cephalosporin antibiotics are delivered directly to the target tissues in a higher concentration than the minimum inhibitory concentration (MIC) that is needed to treat a particular infection while the total systemic dose is a small fraction of the amount needed when given systemically.

The Phase I pilot study (completed) has previously established the MIC for Cefazolin against several strains of MRSA and found the average MIC to be 190μg/ml with the most resistant being 512 μg/ml. Using direct antibiotic delivery, we have successfully treated MRSA cellulitis in numerous patients using Cephalosporin antibiotics in a concentration of one gram in 100 ml of saline (one gram can be given in 2.5 ml of saline IM). Antibiotics that have traditionally been used to treat MRSA infections, such as Vancomycin, can be toxic with potentially severe systemic complications if not monitored closely. Our method of direct antibiotic delivery can be used to deliver a non-toxic Cephalopsorin antibiotic using a small fraction of the amount that is required when giving any antibiotic systemically.

Therapeutic tissue levels have been measured comparing penetration in normal and diabetic patients. As expected, there is a lower tissue penetration in diabetics with traditional methods of delivery. Success of direct antibiotic delivery is completely independent of blood supply and results in much improved outcomes for people with impaired circulation.

The Procedure
The procedure is fast and simple. The patient is put under general anesthesia or conscious sedation for approximately 15 minutes. Cephalosporin solution is then injected with a peristaltic pump (part of the device) using a blunt infusion cannula into the superficial subcutaneous plane of the infected area. The infusion takes less than one minute. Following the infusion, the ultrasound applicator, having been placed in a sterile shield containing ultrasound gel, is passed slowly back and forth over the area of infusion for one to three minutes depending upon the area, resulting in wide dispersion of the antibiotic down to the deep fascia, joint, and bone. All procedures in this study have been performed using the Mettler Surgical ME800 Ultrasound Tissue Preparation System, which is an FDA cleared device for ultrasonic dispersion of infused fluid.

Off-Label Use
During the course of this study, a 79 year-old man who had an MRSA rash required an urgent sternotomy. Because much higher levels of Cefazolin had been demonstrated when given directly using direct antibiotic delivery, and because Cefazolin can be safely injected intramuscularly, Cefazolin was infused under the pre-sternal skin and the antibiotic was dispersed with external ultrasound. The wound healed without incident and the surrounding MRSA rash cleared. These results led to further tests in the microbiology lab where the MRSA was tested against Cefazolin at 1000 μ/ml. The result was complete effectiveness of Cefazolin at this concentration. This information was provided to David P. Nicolau, PharmD FCCP, FIDSA Director of The Center for Anti-Infective Research and Development at The Hartford Hospital, who titrated several very resistant strains of MRSA with controls against various strains of MRSA. The result was a MIC average of 188 μ/ml., with the median of 169 μ/ml., the most resistant being 512 μ/ml.

This dramatic outcome prompted administrators and staff physicians at Santa Rosa Memorial Hospital, Santa Rosa, California, to hold a special meeting with the hospital IRB attorneys to discuss treating patients with severe long-term chronic infections. Dr. Silberg was granted permission to treat patients off label, with patient consent, by the Pharmacy and Nutrition Committee. Subsequently, since June 2011, more than 75 patients have been treated successfully for chronic and acute MRSA cellulitis, many with extensive skin loss, using direct antibiotic delivery (DAD) of Cephalosporins into the infected tissues. This treatment has offered a dramatic and rapid resolution of infections and the ability to successfully split thickness skin graft the areas of skin loss after only a few days. Some of the patients had suffered with wound infections for a number of years. Most patients required only one treatment. No negative side effects have been reported and recurrent infections have been rare.

Case Studies

The following is a sampling of case studies of patients treated for cellulitis and MRSA infections using antibiotic infusion followed by external ultrasound. Between June and December of 2011, more than 75 patients have been successfully treated with this method.

1. A 52 year old patient with chronic MRSA infection. Patient had been treated with IV antibiotics over a period of nine years. Patient received antibiotic infusion and reported significant reduction of pain within a few hours after treatment. A skin graft was applied the same day. Three months post treatment, there has been no recurrence of infection and no negative side effects were reported.


Before Antibiotic Infusion - Infection present for 9 years	After Antibiotic Infusion - 5 weeks post-op

More case studies to follow.

Phase I Clinical Trial - completed

Title
Subcutaneous and Intravenous Tissue Levels of Cefazolin During Abdominoplasty:
A Pilot Study

Investigator
Barry N. Silberg, MD, F.A.C.S. Principal Investigator

Purpose
The purpose of this study is collection and analysis of tissue levels of cefazolin in a cohort of patients undergoing an abdominoplasty as a preliminary to a clinical trial.

Project Objectives
The objective of this proposal is to assess tissue levels of cefazolin following traditional intravenous administration with subcutaneous administration using a tissue dispersion technique in a small sample of patients undergoing elective abdominoplasty.

Specific Aims
Using a sample of adults undergoing elective abdominoplasty the study aim is to:

Compare the dispersion of cefazolin using two different methods of administration

Hypothesis

Tissue cefazolin concentrations are higher when cefazolin is administered subcutaneously using ultrasonic fluid dispersion directly into the surgical site compared to traditional intravenous administration.

Background and Significance
Nearly 30 million patients in the United States undergo surgical procedures annually ¹. Site infection following surgery negatively impacts patients, providers, and the health care system. Postoperative infections contribute to increased hospital stays, delay patients’ return to previous level of physical ability, cause increased pain, and increase the risk of death as a result of sepsis and multi-system organ failure ^1-3. It is estimated that over 600,000 surgical site infections (SSIs) occur annually ¹, with a health care cost of over one billion dollars ⁴. Open abdominal surgery has one of the highest rates of SSI. Recently, Kabon, et al ⁵ reported a prevalence of 9% to 27% incidence of SSI in colon surgery, and Sorenson et al ⁶, reported a rate of 6% to 16% in gastrointestinal surgery. The National Nosocomial Infection Surveillance System (NNIS) reported the incidence of infection for certain abdominal surgeries as between 1.7% and 11.6%¹⁴.

Subcutaneous delivery of antibiotics followed by external ultrasonic dispersion has been adapted from plastic surgery procedures used during liposuction and body contouring and is proposed as a new approach to an old problem. The antibiotic of interest is cefazolin (Ancef, Kefzol) mixed in a specific volume of saline which is introduced into the surgical area just prior to the incision being made, and after the patient has been anesthetized. The fluid is dispersed deeply into the subcutaneous tissue layer by application of external ultrasound for three minutes. The operation then proceeds in the usual fashion. Theoretical constructs underpinning the intervention are delivery of antibiotics directly to the wound site at the time of greatest need and prevention of tissue dehydration during exposure to air to decrease potential tissue necrosis. Dispersing the antibiotic directly into the tissue just prior to incision ensures high local tissue levels at the time when antibiotic protection is most needed.

Tissue and serum analysis compares the cefazolin levels between intravenous and subcutaneous administration. Prophylactic antibiotics are given ideally within 30 minutes of incision to ensure local tissue penetration and protection against infection from common skin bacteria (Fry, 2006). Assuring adequate tissue levels have been problematic, however, as tissue penetration is dependent upon the patient’s perfusion status, vascular state, timing of administration and appropriate dose.

Simultaneous serum and deep subcutaneous tissue samples collected at the completion of the incision and at one hour will yield levels of Cefazolin to compare between intervention and control groups. In general, plasma is spun off from serum and frozen at -40⁰ C until analyzed. Tissue samples are also frozen until analyzed. A buffer is added to weighed, minced samples, which are then centrifuged and decanted for assay ⁷.

Methods

A.	Design: A cross-sectional descriptive design with repeated measures.
B.	Sample Participants will be recruited from adults > 21 years scheduled for elective abdominoplasty procedures at Santa Rosa Memorial Hospital.
C.	Instruments The Silberg Tissue Preparation System ^TM Model ME 800 (9801427) (Mettler Surgical, Anaheim, CA) will be used for subcutaneous saline infusion and application of external ultrasound. The system consists of a 1 Mhz ultrasonic generator device, a 10 cm²ultrasonic applicator, a peristaltic pump irrigation unit, a foot switch, a size 3 French hollow stainless steel cannula, an IV pole, irrigation tubing and a system cart.
D.	Procedure After approval by the Santa Rosa Memorial Hospital Institutional Review Board a convenience sample of patients scheduled for elective abdominoplasty will be screened in the usual manner to determine eligibility for the procedure. The investigator, at the initial consultation, will approach potential subjects preoperatively. Informed written consent will be obtained. Subjects in group A will receive the following intervention: Participants will receive intravenous antibiotic (cefazolin) prophylaxis as ordered by the surgeon in accordance with hospital protocol. Fatty tissue samples will be obtained from fascial level of the wound at the time of incision and at 1 hour or prior to closing, whichever comes first. Simultaneous serum samples will be collected. Subjects in group B will receive the following intervention: Once in the operating suite, and under general anesthesia and sterile conditions, the operative area will be prepped in accordance with standard procedure. A small stab wound will be made with #15 scalpel blade at the end point of the intended incision. The irrigating cannula, previously connected to the peristaltic pump, will be introduced into the subcutaneous tissue through the stab wound. 250 mls of pre-warmed saline containing 1 gram of Cefazolin will be infused approximately 1 cm under the skin as the cannula is advanced along the intended path of the incision. Following infusion, ultrasound energy at 3 watts/cm² will be continuously applied with the ultrasonic applicator for 3 minutes externally to disperse the saline into the surrounding tissue. Fatty tissue samples will be obtained from the level adjacent to deep fascia at the time of incision and at 1 hour or prior to closing, whichever comes first. Simultaneous serum samples will be collected.
E.	Protocols Tissue samples weighing approximately 15 grams each from all subjects will be obtained at the fascial level of the incision immediately after the incision is made and 1 hour later, or at closing, whichever occurs first. Blood samples of 10 mls each will be collected simultaneously. Samples will be frozen at -40^o C and sent to David P. Nicolau, PharmD, FCCP, Center for Anti-Infective Research & Development, Hartford Hospital, Hartford, CT for analysis of antiobiotic levels. All data will be recorded on data specific collection sheets, filed in a locked cabinet kept in a locked room. Data for all participants will be collected on gender, age, admission date, surgery date, discharge date, and readmission within 30 days. Data will include type and length of surgery, length, depth and placement of the incision, anesthetic class, hemoglobin, hematocrit, and antibiotic use beyond the expected prophylaxis.

Statistical Analysis

Frequency distributions and histograms will be used to screen all data for missing values and accuracy of data entry. Data will be analyzed for normality of distribution. Measures of central tendency will be used to characterize the data. Differences in antibiotic tissue and serum levels will be compared through cross tabulation tables, the χ² Test of Independence, and standardized residuals. Within subject means will be compared using t-tests for normally distributed data. Between group means will be compared using analysis of variance and correlation techniques.

Reference List

1.	Bratzler DW, Houck PM, Richards C, et al. Use of antimicrobial prophylaxis for major surgery: baseline results from the National Surgical Infection Prevention Project. Archives of Surgery. Feb 2005;140(2):174-182.
2.	Mangram AJ, Horan TC, Pearson ML, Silver LC, Jarvis WR. Guideline for prevention of surgical site infection. Infection Control & Hospital Epidemiology. 1999;20:247-280.
3.	Nichols RL. Preventing Surgical Site Infections: A Surgeon's Perspective. Centers for Disease Control: Emerging Infectious Diseases. 2001;7(2).
4.	Kaye KS, Schmit K, Pieper C, et al. The effect of increasing age on the risk of surgical site infection. J Infect Dis. Apr 1 2005;191(7):1056-1062.
5.	Kabon B, Akca O, Taguchi A, et al. Supplemental intravenous crystalloid administration does not reduce the risk of surgical wound infection. Anesth Analg. Nov 2005;101(5):1546-1553.
6.	Sorensen LT, Hemmingsen U, Kallehave F, et al. Risk factors for tissue and wound complications in gastrointestinal surgery. Ann Surg. Apr 2005;241(4):654-658.
7.	Dudley MN, Nightingale CH, Drezner AD, Low HB, Quintiliani R. Comparative penetration of cefonicid and cefazolin into the atrial appendage and pericardial fluid of patients undergoing open-heart surgery. Antimicrob Agents Chemother. Sep 1984;26(3):347-350.

Results of Phase I Clinical Trials

Effects on MSRA

Cefazolin administered intravenously generates adipose tissue levels between 3-5 micrograms/ml after injection. This is far below therapeutic tissue levels. Using the Silberg Tissue Preparation System in our IRB study, tissue levels at the time of surgery average 1200 micrograms/ml in the target area.

Clinical testing for the effectiveness of Cefazolin when used against MRSA utilizes concentrations no greater than 250 micrograms/ml. At this dose Cefazolin is ineffective against MRSA.

The two slides demonstrate the effectiveness of Cefazolin at a concentration of 1000 micrograms/ml when used against MRSA. The first slide is at 20 hours and the second is at 40 hours.


At 20 Hours	At 40 Hours