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

The U.S. media has given a lot of attention to H1N1 A Virus since the intial announced case in April 2009.  According to a recent article in the October issue of Infection Control Today, there are several mechanisms of transmission for influenza.  First of all, it comes through close contact between persons.  In several studies, close contact has been defined as three feet or closer.  Some studies say transmission can occur within six feet.

The mechanisms of transmission are:

1. Exposure to large respiratory droplets
2. Exposure to small-particle aerosols in the immediate vicinity
3. Direct contact transfer from contaminated hands to the nose and eyes

There is no proven evidence that shows one mechanism is more effectiven than the other.  Droplet transmission requires close contact and is generated by the infected person sneezing, coughing or talking.  Localized airborne transmission of influenza via small particles may occur in short distances (as much as 6 feet).   Influenza is not known to spread in airborne transmission of distances any longer than this, thus special air handling systems are not necessary to prevent the spread of influenza in healthcare settings.  Direct contact transmission of influenza has been suggested as a contributing factor in several studies.  It is therefore important to practice thorough and frequent handwashing as well as masks, gloves and gowns in a healthcare environment.

Are you practicing good techniques in your work place, in your home and in any place you come in contact with others to prevent transmission of the H1N1 A virus?  Does your employer have an H1N1 A virus prevention or response program should an outbreak occur in your company?

 – Jim Verzuh, Director of Marketing, BioScience Laboratories

To date, the mechanisms of virus inactivation by microbicides are not well understood. Several modes of inactivation have been studied quite superficially. High temperature treatment is known to induce major changes in quaternary protein structures, resulting in a loss of continuity of the viral capsid icosahedral geometry. Hypochlorite is considered an oxidizing and cross-linking agent that causes viral protein disintegration. Ultraviolet (UV), supposedly, targets a small portion of a protein molecule containing vulnerable amino acids with high photochemical lability to UV.  On the molecular level, the mechanism of inactivation was first studied with representatives of the genus Enterovirus. It was shown that structural differences in the VP1 region of these viruses are crucial for inactivation with glutaraldehyde. Sensitive and moderately sensitive enteroviruses exhibited lysine residues in the outermost DE and BC loops of VP1 major capsid protein, while resistant viruses had no lysine residues in the N-terminus, β-strand D, BC, DE, and G2H loops of VP1. The identity in the VP1 region of MNV and human NoV is less than 50% which suggests the possibility of substantial discrepancies in their sensitivity to inactivation. The discrepancies observed among seven genetically closely related enteroviruses raises concern that testing results obtained using surrogate pathogens, even closely related, may not always be adequately extrapolative to human viruses.

Volha  Dzyakanava, Ph.D., Manager of the Virology Laboratory

 Historically, the effectiveness of virus inactivation is estimated using procedures for detecting the loss of the virus’ ability to replicate. Until recently, this approach could not be applied to the majority of noncultivable viruses. There have been several attempts to develop indirect infectivity assays in order to solve the problem. One attempt is the enzymatic pretreatment of inactivated viruses with proteinase K and Rnase . Different research groups presented contradictory results on the reliability of this method. For the most part, it is because the effectiveness of the enzymatic pretreatment depends on the type of inactivation producing different levels of disruption, and hence, different levels of accessibility of peptide bonds to proteinase. Moreover, enzymatic pretreatment represents a secondary inactivation of presumably inactivated viruses, which might further enhance virus disruption.  Relatively reliable results were obtained by assessing loss of the inactivated virus’ ability to bind cellular receptors. The cell attachment capabilities of Poliovirus, Feline Calicivirus and HAV were lost in response to high temperature and hypochlorite treatment. However, UV inactivation did not prevent HAV from binding to the cellular receptor. This indicates that alterations of virus cell-binding sites are not the only mechanism of inactivation.   As a whole, detection of virus viability remains essential. The infectivity assay by means of cell culture has been the gold standard against which all new technologies for virus detection are evaluated. Methodologies combining cell culture and molecular techniques – for example, ICC-PCR (Integrated with Cell Culture PCR) and its modifications – have been used successfully for rapid detection of infectious enteroviruses and adenoviruses in environmental samples. The ICC-PCR technique is based on PCR analysis of the cell culture after incubation with test samples. Cell culture incubation allows elimination of non-infectious viruses and multiplication of infectious viruses, which then are detected by PCR. This method provides rapid and reliable detection of virus viability. Although ICC-PCR was developed originally for the detection of infectious viruses in environmental samples, the general principle of this method is applicable to detection of a replicating viruses in cells – for instance, the strand-specific PCR detection of Hepatitis C virus (HCV) in peripheral blood mononuclear cells. In this application, viral replication in cells is detected in terms of a HCV replicative intermediate, which is a negative-strand RNA. Detection on the basis of the negative strand means that false positive results due to amplification of RNAs of non-replicating viruses are avoided. Generally ICC-PCR is a very promising approach which with some modifications can be applied to identification of virus inactivation.

Volha Dzyakanava, PhD – Manager Virology Laboratory

Several animal caliciviruses are known to be genetically closely related to human Norovirus, but only two have been studied for use as surrogates. These viruses are Feline Calicivirus (FCV) and Murine Norovirus (MNV).  FCV, a member of Vesivirus genus, can be readily propagated in cell cultures and has been extensively used as a surrogate for microbicide efficacy testing. As a respiratory pathogen, FCV is relatively sensitive to low pH treatment, which might represent a significant difference from the enteric human virus. MNV is the first cultivable animal virus representative of the Norovirus genus. This pathogen infects a wide range of laboratory mice and, like the human virus, is transmitted via the fecal-oral route. Nevertheless, MNV causes multiple clinical presentations in their natural host that are very different from those of human Norovirus. Despite the lack of experimental data on MNV and human Norovirus correlation, the murine pathogen is currently considered the most relevant surrogate, which means that manufacturers of microbicidal agents registered in the past based on FCV-testing will probably be required  to re-test against MNV.

New types of calicivirus are being discovered in human and animal populations, and some may have potential as surrogates. One example is the Tulane virus recently isolated from rhesus macaques, a host much closer evolutionarily to humans. It has been shown to exhibit the highest homology scores with the representatives of genus Norovirus in amino acid sequences of NTPase, Polymerase, and VP1 regions. From a practical point of view, establishment of the adequate surrogate will prevent recurrent evaluation with every newly discovered, but not experimentally validated virus, as well as undue financial pressure on manufacturers.

Potentially, the use of the closely related caliciviruses as surrogates might not be necessary. Viruses such as Poliovirus and Hepatitis A virus have been extensively studied for over five decades, and both are human enteric pathogens. Because of their evolutionary adaptation, the human enteric viruses may be more relevant with regard to chemical tolerance or environmental resistance than are viruses originated from distant hosts.