BioScience Laboratories, Inc., personnel participate in the activities of numerous national and international professional associations that focus on microbiology and infection control in the healthcare and food service industries.  As our website indicates, our interests relate to disinfectant and topical antimicrobial formulations, their importance in reducing the risk of disease transmission, and fair assessments of their antimicrobial efficacy.  Because such assessments require methods of testing that provide reliably reproducible data meaningful in the context of infection control, our personnel have, for many years, been deeply involved in method development through the American Association for Testing and Materials (ASTM), specifically, Subcommittee E 35.15 on Antimicrobials.  Four members of our staff, including myself as Subcommittee Co-Chair, serve on E 35.15.

As of the conclusion of our semiannual meeting last week, our Subcommittee has 96 members and is responsible for 45 approved methods, plus another 13 currently in the process of development.  In the interest of brevity, I will describe only two examples of the latter.

The first of special note is a modification of E 1174, the ASTM version of the FDA method specified for testing of handwash products intended for use in healthcare. The modification involves the procedure for contaminating the hands with Serratia marcescens, the indicator bacterium used to challenge product antimicrobial efficacy, and is particularly important in that the new method will be much more appropriate for testing leave-on (non-water-aided) hand sanitizers.

Another method-in-the-making is one for testing liquid microbicides versus bacterial biofilms, organized assemblages that are considerably more resistant to antibiotics, topical antimicrobials, and disinfectants than are planktonic (free-floating) bacteria.  Only in the last decade, or so, has the important role that biofilms play in disease causation and environmental fouling been understood, and colleagues from the Center for Biofilm Engineering at Montana State University here in Bozeman have been in the forefront of methods development in E 35.15.

Although I have selected for comment only these from among our many methods, I would welcome any questions you may have about testing of antimicrobial formulations and how the testing methods are created collaboratively by volunteers from industry, regulatory agencies, and CROs such as BioScience Laboratories.

– John Mitchell, Director of Quality Assurance and Chief Medical Officer

We have been very fortunate in the past regarding results from FDA, EPA, and Sponsor audits.  We take each one seriously, as a learning experience, and implement appropriate changes to our processes in a timely fashion.  The audits are a constant reminder for BioScience to strive to be the best that we can be.  Although we are not ISO 17025 certified, we have been audited using those guidelines, as well as Good Laboratory Practice Regulations (GLPs) and Good Clinical Practice Regulations (GCPs).  Some outcomes of the audits have included: adding “Controlled” and “Uncontrolled” stamps on copies given to auditors; adding “Obsolete” on documents once a revision has been put into place; and being more diligent in referencing other SOPs in our SOP documents.  We are accommodating in sending, prior to an auditor’s visit, such information as our current organizational chart, corporate resumes for key employees, and the index of our Standard Operating Procedures.  We have received praise on the ease with which we are able to access documentation requested by an auditor.  All in all, each audit has been a positive experience for us, and I believe for the auditor as well.  Come visit us and see for yourself.  Scientific Expertise with Montana Hospitality is a self-appraisal we take very seriously.

Amy L. Juhnke, Manager of Quality Assurance/Document Control

For those wondering about the responsibilities of a principal investigator, here is the result of my research on this topic.

The Principal Investigator receives no direct supervision and has complete authority and responsibility for the clinical trials they perform. Personal initiative is imperative. He or she must have a firm understanding of the principles of planning, organizing and management. The Principal Investigator must promote Good Clinical Practices thought the clinical research process by ensuring strict adherence to the protocol, protection of the patient’s health and welfare, integrity of the data generated, and compliance with all federal and state regulations The person who holds this position must have a current physician’s licensure to practice with five to eight years of experience in clinical research. They must have an above average ability to interface with patients, staff, sponsor, and IRB. The Principal Investigator is responsible for maintaining a cohesive team that consistently completes clinical trials on time and at budget.

Provides investigator qualifications and agreements to sponsors by maintaining current curriculum vitae, demonstrates the proper education, training and experience to conduct the clinical investigations Principal Investigator is responsible for signing Form FDA 1572, the protocol, and all sponsor contract(s), and complete disclose of any conflicts of interest

Ensures protocol compliance by possesses a thorough understanding of the protocol, determines if the inclusion/exclusion criteria are compatible with the intended study population, establishes recruitment goals and gains approval of both the IRB and the sponsor prior to any amendments or protocol deviations

Ensures continuous oversight of the clinical trial by the IRB, by providing the protocol, investigator’s brochure, informed consent, recruitment materials, protocol amendments or deviations, adverse events and any other materials regarding the study to the IRB for review

Ensures adequate facility and number of staff to conduct the study and makes adequate time to supervise and conduct the study.

Manages medical care and ensures that all medical decisions related to the clinical trial are made by a qualified physician. The Principal Investigator evaluates subject compliance and response to therapy, is responsible for adverse events, proper medical attention in the occurrence of an adverse event, and notification of primary care physician

Protects the rights and welfare of subjects ensuring the informed consent is in accordance with CFG 56 and 45 and that the subjects are aware of all aspects of the clinical trial

Oversees the quality of the trial by ensuring all case report forms are legible, complete, and accurately reflect the source documents. Reviews, understands, and approves corrections made to case report forms and ensures the proper quarantine and use of the investigational materials

Communicates effectively with subjects, the research team, the sponsor, and the IRB and is responsible for the clarity of the protocol and purpose of therapies by the research staff. The Principal Investigator will participate in study monitoring conducted by the sponsor and regulatory administrations

The Principal Investigator maintains professional knowledge and technical expertise by reviewing medical research publications and participating in professional organizations

John Dyba, Senior Account Executive

As a clinical research manager conducting research on a vulnerable population particular attention must be given to the process of informed consent. This additional attention is required because vulnerable populations are populations who have limitations regarding their ability to provide informed consent and are more susceptible to coercion or undue influence. Because the decision to participate in clinical research must be made by a competent individual who has received the necessary information, and who has understood that information, special care must be taken to resolve language and comprehension barriers between the researchers and their potential subjects.

Therefore the researchers must set aside adequate time and resources to insure that the proper language, or languages, is used to inform the potential subjects of the requirements of informed consent. Not only should the language itself be presented at a comprehension level easily understood, but the content and concepts of the message must also be presented at a level that will allow their comprehension. Particular attention should be given to the concepts of clinical research, randomization, and inactive medications (placebo) due to complexity of the issues, their importance, and the relative inability of the normal population to completely comprehend them.

Each of these issues requires the attention of the Institutional Review Board (IRB). It is the responsibility of the IRB to determine that the minimal requirements of 45 CFR 46.111 are achieved, that additional safeguards for this vulnerable population are in place and that proper informed consent is obtained from each subject, prior to protocol approval.

Private, for-profit IRBs can function without ethical conflicts as long as their members are not directly involved in the research and the outcome of the review in no way benefits them as individuals.

John Dyba, Senior Account Executive

In the Clinical Laboratory, we employ numerous methods to sample microorganisms from the skin of human volunteers. These sampling methods are not normally part of a microbiologist’s college curriculum, and newly hired technicians need to be trained appropriately to perform the methods in our laboratories. Two of the methods primarily employed at BioScience Laboratories, Inc. (BSLI) are the Glove-Juice Sampling Procedure and the Cylinder-Sampling Technique (also sometimes call the Cup-Scrub Method). In addition to proper training, we also employ statistical techniques to help identify those requiring additional training and for improving upon our methods.

For studies that involve evaluating Handwash products, we employ the Glove-Juice Sampling Procedure to sample microorganisms from the hands. A sterile glove is placed over the hand and a measured volume of sampling fluid is placed in the glove. The method of gloving can differ depending on the type of study. Hands may be wet, dry, held higher than elbow, or held lower than elbow. The glove is secured at the wrist using a tight-fitting elastic band. The hand is then massaged in a standardized manner for 1 minute by the technician. Following the massage, a sterile pipette is used to draw out a volume of fluid that is then diluted and plated. As you can see, the multitude of steps would have to be practiced and standardized so that all technicians perform the procedure in the same manner, and mistakes that may cause the loss of data are not made.

Sampling microorganisms from a small area of skin is performed using the Cylinder-Sampling Technique. A small (~1 in diameter), stainless steel cylinder is held tightly on the skin with one hand, while the other hand uses a pipette to place a small measured volume of fluid into the cylinder. A rubber-tipped glass rod is then rubbed against the skin inside the cylinder for 1 minute using a standardized, sweeping motion. A new pipette is used to transfer the fluid to a test tube. The procedure is repeated, with the second volume of fluid being removed and pooled with that fluid from the first sample. In addition to being trained to perform the procedure, technicians also need to learn the importance of cylinder placement and how to deal with sampling at a multitude of anatomical sites. This technique, too, requires lengthy training and practice.

Over my tenure at BSLI, Dr. Paulson has always employed statistical methods, such as Exploratory Data Analysis, to “look” at the data to determine and identify data points that are extreme “outliers” compared to the other data points. This statistical technique is used to identify human volunteers who may have not followed product restrictions or those who did not perform a wash procedure correctly, as well as help to determine if a noted incident that occurred had an affect on the data. EDA can also be used to evaluate the sampling methods and the technicians performing them… that is, identifying technicians who may require additional training. Conversely, EDA can also be used to identify technicians who perform the procedure well. The sampling method then can be adjusted to how they perform it, thereby decreasing variability and increasing the acuity of the conclusions drawn from the data.

Properly training technicians at BioScience Laboratories, Inc., is always a priority. We understand that our methods are non-traditional, but data generated by them is important to evaluating the true efficacy of a product. The initial training of a technician is vital to the conductance of the study, but continually monitoring the technicians’ performance and identifying minor adjustments has been and always will be a continual process here at BioScience Laboratories, Inc.

Christopher M. Beausoleil, CCRP

MRSA is a resistant variation of the common bacterium Staphylococcus aureus. It has evolved an ability to survive treatment with most antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for difficult-to-treat infections in humans.

The organism is often sub-categorized as Community-Associated MRSA (CA-MRSA) or Health Care-Associated MRSA (HA-MRSA) although this distinction is complex. Some have defined CA-MRSA by criteria related to patients suffering from an MRSA infection while other authors have defined CA-MRSA by genetic characteristics of the bacteria themselves. The new CA-MRSA strains have rapidly spread in the United States to become the most common cause of cultured skin infections among individuals seeking medical care for these infections at emergency rooms in cities. These strains also commonly cause skin infections in athletes, jail and prison detainees, and soldiers.

Solution:
The overwhelming response in regards to the management and reduction in infection rates of these two pathogenic organisms were very basic; wash your hands regularly with an antimicrobial product and disinfect surfaces with a hard surface disinfectant–simple solutions to a very big problem.

BioScience Laboratories, Inc. has the capability of testing antimicrobial products, both topically applied antiseptics and hard surface disinfectants, for their efficacy against these organisms. To determine if a product is effective in killing these bacteria, standard in-vitro evaluations can be used to determine their efficacy. To take it a step further, one can evaluate their products’ efficacy in an IN-USE setting; that is, surrogate clinical trials on human volunteers. BSLI has recently been approved to conduct efficacy evaluations against C. diff and MRSA on Human Subjects. By working with an Institutional Review Board, BSLI has developed a Protocol to test the efficacy of topical antiseptic products on the skin. We feel that this type of testing will aid in the development of new novel antimicrobial products that can be used in day to day life as well as in hospital settings to help protect lives and reduce infections rates worldwide.

Kyle McGovern
Senior Account Executive

Most of the methods employed by BioScience Laboratories, Inc. to evaluate antimicrobial efficacy of products involves quantitative techniques such as microbial counts.  The accuracy of these methods heavily depends upon inhibiting or neutralizing the antimicrobial activity of the product at specified times following exposure.  Recently, manufacturers of antimicrobial products are developing compounds or mixtures of compounds that have required additional attention to the inactivation of the antimicrobial activity of the product.  Publications have addressed how to perform and validate these neutralization procedures (see references).  

How does an investigator first decide on an appropriate method for inhibiting the antimicrobial activity of the product; in other words, what neutralizer system should be employed in efficacy testing?  Investigators have three basic neutralizer systems available to them; diluting to sub-inhibitory levels, chemical inactivation, or physically separating the product from the microorganism.  In some cases, a combination of these methods may be required to achieve inactivation.

Alcohol products are the most common type immediately neutralized by dilution.  Other products may require greater dilution before the inhibitory effects are eliminated.  In such situations, a chemical inactivator(s) can aid in reducing the dilution ratio required, and thereby, increasing the detection level of the product-testing for efficacy.  Dilution of the product may also be achieved, de facto, by performing an In Vivo neutralization assay (for those efficacy evaluations involving human volunteers).  In Vivo assays employs the application procedure to be used in the evaluation and the amount of product to be applied by the human volunteer.  Because the amount of product remaining on the skin of the subject post-application is much smaller than the volume of product used in an in-vitro neutralization procedure, the effects of neutralization by dilution are much greater.

Chemical inactivation is most often appropriate for neutralization of products that contain actives that are cationic or anionic in their activity.  Chemical compounds that have a stronger affinity for the active than does the target microorganism, but are themselves non-inhibitory, allow for design of a relatively simple neutralizer system. 

When dilution to sub-inhibitory levels and/or chemical inactivation fail to neutralize a product, physically separating the microorganism from the product is another, although cumbersome, alternative that requires use of a membrane filtration unit.  Diluting the product and using chemical inactivators as a rinsing solution can also be combined with membrane filtration.   Although membrane filtration in any combination is the most costly and time-consuming of the methods, it may be one’s only choice for certain antimicrobially active compounds. 

When performing antimicrobial efficacy evaluations, sponsors and investigators need to be made aware of the importance of determining a neutralizer system and the validation of the system.  Failure to neutralize the antimicrobial activity of a product will produce results that cause a sponsor to conclude that a product is efficacious when it is not.   We, here at BioScience Laboratories, Inc. have years of experience developing and validating systems for neutralizing a wide variety of antimicrobial products.  We fully understand the importance of producing results that demonstrate the actual efficacy of an antimicrobial product, and look forward to helping our clients in this endeavor. 

Christopher M. Beausoleil, CCRP

Senior Clinical Director

References

Sutton, Scott V.W. 1996. Neutralizer Evaluations as Control Experiments for Antimicrobial Efficacy Tests.  In: Handbook of Disinfectants and Antiseptics, J.M. Ascenzi, Ed. Marcel Dekker, New York. 300pp.

ASTM E 1054-08, Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents, ASTM International, 100 Barr Harbor Drive, P.O. Box C700, West Conshohocken, PA 19428-2959, United States.

Beausoleil, Christopher M.  2003.  A Guide for Validation of Neutralizer Systems Used in Topical Antimicrobial Evaluations.  In: Handbook of Topical Antimicrobials, D.S. Paulson, Ed. Marcel Dekker, New York. 452 pp.