reading

Chapter 2: Hazard Criteria and Categorization of Microbes Classification Systems

Chapter 4: Animal Biosafety Level 3 Facility – Enhancements When Dealing with Large Animals

4.1 Enhancements to Upgrade a Standard Animal BSL-3 Facility to a LABSL-3 Facility Housing Large Animals

6.6 Packing Instruction 620 for Infectious Substance, Category A; UN 2814 and UN 2900

Chapter 10: Bridging the Gap between Requirements of Biocontainmentand Diagnostics

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty can be created or extended by sales representatives or written sales materials. The Advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.

Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley's global Scientific, Technical, and Medical business with Blackwell Publishing.

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form — by photoprinting, microfilm, or any other means — nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Acknowledgement

This project has been funded with support from the European Commission. This publication reflects the views only of the author, and the European Commission cannot be held responsible for any use which may be made of the information contained therein.

With the financial support of the Prevention of and Fight against Crime ProgrammeEuropean Commission – Directorate-General Home Affairs

Preface

This is the fourth book written in a series that started within the framework of the European project COST (European Cooperation in Science and Technology) action B28, which aimed at increasing knowledge on BSL-3 (biosafety level) and BSL-4 agents; to support development of more accurate diagnostic assays, vaccines, and therapeutics; and to better understand epidemiology of these highly pathogenic microorganisms that can potentially be used as biological weapons. COST funding has ended in 2010 and an additional grant by DG Home Affairs in the program “Prevention of and fight against crime” now sponsors the final volume “A practical guideline to working in BSL-3/4 laboratories.”

The first book summarized the knowledge on microarray technology. The second book summarized the knowledge on proteomics, glycomics, and antigenicity of the BSL-3/4 agents. The third book described the agents themselves.

The authors of the chapters are all involved in research concerning these agents and have been working with them extensively. Typically, the authors also have access to BSL-3/4 laboratories in which they can work with these agents, thus are familiar with the required precautions and legislations. Their expertise has also been employed for assessment of outbreaks and understanding the epidemiological factors that facilitate their spread and subsequent control.

This book was created from lectures given in a training school course held four times funded by COST action from 2007 to 2010 and four times funded by DG Home in 2012–2013. At the time of publication, two more courses have to be held.

List of Contributors

Francesc Xavier Abad

CReSA

Centre de Recerca en Sanitat Animal

UAB-IRTA

Bellaterra

08193 Barcelona

Spain

Åsa S. Björndal

Swedish Institute for Communicable Disease Control

Unit for Biorisk Management

Department för Analysis and Prevention

Nobels väg 18

SE-17182 Solna

Sweden

Patrick Butaye

Veterinary and Agrochemical Research Centre

Department of Bacteriology and Immunology

Groeselenberg 99

1180 Brussels

Belgium

Mariano Domingo

CReSA

Centre de Recerca en Sanitat Animal

UAB-IRTA

Bellaterra

08193 Barcelona

Spain

Mandy Elschner

Friedrich-Loeffler-Institut

Federal Research Institute for Animal Health

Institute of Bacterial Infections and Zoonoses

Naumburger Straße 96a

07743 Jena

Germany

Martin Heller

Friedrich-Loeffler-Institut

Federal Research Institute for Animal Health

Institute of Molecular Pathogenesis

Naumburger Straße 96a

07743 Jena

Germany

Frank T. Hufert

University Medical Center Göttingen

Department of Virology

Kreuzbergring 57

37075 Göttingen

Germany

Annette A. Kraus

Swedish Institute for Communicable Disease Control (SMI)

Department for Diagnostics and Vaccinology

Nobels väg 18

SE-17182 Solna

Sweden

Jürgen Mertsching

Hannover Medical School

Department of Biological Safety

Carl-Neuberg-Straße 1

30625 Hannover

Germany

Ali Mirazimi

Swedish Institute for Communicable Disease Control (SMI)

Department for Diagnostics and Vaccinology

Nobels väg 18

SE-171 82 Solna

Sweden

Nigel Silman

Research & Development

Public Health England

Porton Down

SP4 0JG Salisbury

United Kingdom

David Solanes

CReSA

Centre de Recerca en Sanitat Animal

UAB-IRTA

Bellaterra

08193 Barcelona

Spain

Manfred Weidmann

University Medical Center Göttingen

Department of Virology

Kreuzbergring 57

37075 Göttingen

Germany

Introduction

Biosecurity practices cannot be built without a strong safety culture; therefore, it is generally agreed that training for BSL-3/4 (biosafety level) work should be a precondition for starting to work in these specialized, safety and security sensitive, laboratories. Training for work in BSL-3/4 laboratories, however, is hardly available on an organized level and mainly performed in a traditional laboratory culture of individual training. In recent years, however, the demand for broader training of ever more young scientists working in newly created BSL-3/4 laboratories has increased (at the latest count, there were over 600 BSL-3 laboratories in the United States) [1]. The steady rise of people working in this type of laboratory environment may inflate the potential for accidents, as has been seen in the United States in recent years. Therefore, organized training, as an addition to the individual training, is absolutely necessary and indeed has been announced as obligatory in the United States [2]. In Europe, there are several projects offering training, for example, Euronet-P4 has provided additional practical training for BSL-4 laboratory staff, ETIDE has provided training for clinicians for infectious disease emergencies since 2007, and Biosafety Europe has provided safety guidelines [3].

Participants of the COST Action B28 “Array technologies for BSL-3 and BSL-4 pathogens” [4] developed a new training course for scientists working in BSL-3/4 laboratories and ran it once a year from 2007 to 2010. Funding by DG Home Affairs allows for two courses a year since 2012 [5].

The 4-day course consists of lectures and practical training. The background of the lecturers represents all the very different possibilities of organizing a BSL-3/4 laboratory including the adaptation to the local requirements of biosafety, safety at work, and social regulations.

Working in a BSL-3 laboratory is a very good basis to commence work at BSL-4 level, especially, as the danger of infection in a BSL-3 level (a biosafety shell to protect the environment) can be considered to be potentially much higher than that in a BSL-4 laboratory, which offers superior personal protection. The BSL-3 facilities at the Department of Virology, Göttingen University Medicine allow the simultaneous training of 10 students, two each working in one class-2 biological safety cabinet supervised by one lecturer. As training in a BSL-4 at course level of this size is impossible due to security and infrastructure reasons, this course provides at least a good basic practical introduction to the principles of working at BSL-3 that are also adhered to in a BSL-4 environment.

The lectures summarized in this book cover biocontainment, hazard criteria and categorization of microbes, technical specifications of BSL-3 and ABSL-3 (animal biosafety level) laboratories, personal protective gear, shipping BSL-3 and BSL-4 organisms according to UN and IATA (International Air Transport Association) regulations, efficacy of inactivation procedures, fumigation, learning from a history of laboratory accidents, handling samples that arrive for diagnostic testing, and bridging the gap between the requirements of biocontainment and diagnostics. The practical sessions of this course are run over three afternoons and cover the use of personal safety equipment including the use of positive pressure masks, dexterity training, and inactivation procedures for viruses and bacteria [6]. Altogether, the combination of lectures and practicals provides a good focused introduction into principles and regulations important for both types of laboratory environments.

A general conflict between laboratory scientists and biosafety officers in all countries appears to be the difficulty of convincing biosafety officers to accept proof of biosafety that has not yet been cast into official rules and regulations. On the other hand, it is quite clear that regulations cannot cover all aspects of working in these laboratories especially when dealing with new organisms. The SARS-CoV (severe acute respiratory syndrome coronavirus) outbreak in 2003 can be seen as a showcase for this dilemma [7].

Biosafety regulations issued are often not flexible enough and can tend to impede work rather than to increase safety. The notion that the scientists at work cannot be trusted seems contradictory to the fact that the scientists, working with highly contagious and pathogenic agents, have an eminent interest in their own safety and health.

A major effort across the European Union therefore should be to build a flexible framework to accept biosafety evidence created locally by the different respective laboratories. There is a need for a consensus between biosafety organizations and the scientists at work, on how proof of biosafety should be shown and documented in order to sustain and not stifle a flexible capacity to deal with novel and well-known pathogens.

Tighter regulations issued by national biosafety bodies in response to the green paper launched by the European Commission [8] may blur or bias their perception of biosafety necessities, which might make working in BSL-3/4 laboratories close to impossible. It would be ironic if a measure initiated because of political biothreat considerations would essentially impede all biopreparedness actions toward unexpected events in the public domain.

Recently, Kimman et al. [7] have provided a literature review on laboratory-acquired infections (LAIs) including those that occurred in the wake of the SARS-CoV outbreak. Their conclusion was that deviation from general “good microbiological practice” is the most frequent cause for LAI and that training for compliance to procedures and regulations appears to be the best method to avoid these. In this, the lectures summarized here offer the opportunity to improve the available basic training for BSL-3/4 scientists and prerequisite initial training for beginners.

References

1. Gronvall, G.K., Fitzgerald, J., Chamberlain, A., Inglesby, T.V., and O'Toole, T. (2007) High-containment biodefense research laboratories: meeting report and center recommendations. Biosecur. Bioterror., 5, 75–85.

2. Le Duc, J.W., Anderson, K., Bloom, M.E. et al. (2008) Framework for leadership and training of biosafety level 4 laboratory workers. Emerging Infect. Dis., 14, 1685–88.

3. Ippolito, G., Nisii, C., and Capobianchi, M.R. (2008) Networking for infectious-disease emergencies in Europe. Nat. Rev. Microbiol., 6, 564.

4. COST B28 http://www.cost-b28.be/index.php/pages/index (accessed 13 April 2013).

5. Weidmann, M., Hufert, F., Elschner, M. et al. (2009) Networking for BSL-3/4 laboratory scientist training. Nat. Rev. Microbiol., 7, 756.

6. Abteilung Virologie http://www.virologie.uni-goettingen.de/index.php?page=22&empty=1&id=26 (accessed 13 April 2013).

7. Kimman, T.G., Smit, E., and Klein, M.R. (2008) Evidence-based biosafety: a review of the principles and effectiveness of microbiological containment measures. Clin. Microbiol. Rev., 21, 403–25.

Chapter 1

Laboratory Biosafety in Containment Laboratories

Microbiological research on and diagnostics of highly pathogenic microorganisms, namely bacteria and viruses, have to be conducted in containment laboratories in order to contain the infectious material. We are therefore referring to the “Concept of Biocontainment” – a concept, which dates back to the 1940s, when the first biosafety cabinet (BSC) class III was developed at the US Army Biological Warfare Laboratories in Fort Detrick, Maryland.

Biocontainment is required to prevent accidental infection of researchers or diagnostic staff and to avoid release of the infectious agents into the surrounding environment.

1.1 Routes of Infection

Depending on the nature of the microorganism, there is a great variety of infection routes. However, the natural route of transmission may be different in a laboratory setting when working with isolated pathogens. This has to be considered when establishing working procedures for biosafety laboratories.

In a research laboratory, for instance, HIV or hepatitis B virus is not transmitted via the natural route, that is, from person to person through direct contact of body fluids, but, for example, through accidental inoculation with a syringe. The bacterial pathogen Neisseria gonorrhoeae spreads through direct contact under normal circumstances and laboratory workers have also to primarily protect themselves from direct contact (Chapter 10).

Bacteria and viruses that are vector-borne such as the tick-borne encephalitis virus (TBE) or the tick-borne Borrelia sp. obviously have a different infection route in the laboratory as compared to the natural setting. Here, protection should also aim at preventing needlestick injuries with contaminated syringes and direct contact with fluids that have a high concentration of the infectious agent.

While some other bacteria such as Salmonella or Vibrio cholerae spread via the fecal-oral route through contaminated food or water and are relatively easy to contain, others spread readily via aerosols and are more difficult to control. Consequently, when working with bacteria such as Mycobacterium tuberculosis, laboratory workers have to protect themselves by wearing personal protection devices (respirators) and perform the work in BSCs. The same measures have to be taken when working with viral pathogens that also spread through aerosols, such as the avian influenza virus or hantaviruses.

In general, airborne or aerosol-transmitted pathogens are comparatively difficult to work with. Aerosols are practically invisible to the human eye, not to mention airborne viruses or bacteria. Using syringes, pipettes, and mixing devices, even according to good laboratory practice (GLP) protocols, creates aerosols in unexpected amounts. Dimmick et al. conducted a study in the early 1970s and estimated the aerosol dose originating from pipetting 1 ml to be 10¹⁰ small particles. Even at a distance of 3 m, the number of small particles still reaches 50. Depending on the nature of the pathogen, this can clearly reach or exceed the infectious dose of that particular virus or bacterium.

Generally, different precautionary measures have to be applied when working with different pathogens according to the routes of transmission.

1.2 Classification of Microorganisms

When establishing routines for microbiological laboratories, not only the route of infection of the used pathogens has to be considered, but the infectious dose, available countermeasures, and preexisting immunity have to be considered as well. In addition, information about concentration of the isolated pathogen, total volumes used in a certain research or diagnostic setting, as well as experience is important when defining the risk.

While airborne viruses belong to the most difficult pathogens to contain, work with varicella virus, for instance, can be performed under moderate safety precautions, because the disease is treatable and a vaccine is available. HIV, on the other hand, causes a lethal disease that can be neither treated nor prevented by a vaccine, is a rather unstable virus with a comparatively low infectious dose, and can therefore also be handled with moderate safety precautions.

The obvious question is now to classify the microorganisms and translate this classification into levels of precautionary measures, that is, into biological safety levels (BSLs). The levels of containment range from the lowest safety level 1 to the highest at level 4. In the United States, the Centers for Disease Control and Prevention (CDC) have specified these levels. In the European Union, the same BSLs are defined in a directive (Commission Directive 97/65/EC). In summary, the classification of microorganisms is based on various parameters specific for every pathogen including routes of transmission, severity of the disease, infectious dose, available countermeasures or preventive measures, and transmissibility to the community. These may be influenced by existing levels of immunity, density and movement of host population presence of appropriate vectors, and standards of environmental hygiene.

The WHO (World Health Organization) has classified infectious microorganisms by risk groups and the following list provides an overview of the risk levels when working with different pathogens. There are however other classification schemes (Chapter 2):

WHO risk group 1 – minimal risk

Microorganisms that usually do not cause human disease, such as Escherichia coli K12 or Lactobacillus.

WHO risk group 2 – moderate risk

Microorganisms that cause treatable or self-healing diseases and are difficult to contract via aerosol in a laboratory setting, such as salmonella or measles virus.

WHO risk group 3 – high risk

Highly contagious microorganisms that cause serious diseases, such as TBE virus or M. tuberculosis.

WHO risk group 4 – very high risk

Highly contagious microorganisms that cause serious diseases, even epidemics, with high mortality rate, such as Ebola virus or Lassa fever virus.

1.3 General Containment Principles

In general, there are two different levels of protection against accidental infections when working with pathogens in a research or diagnostic laboratory, a primary and a secondary barrier. In addition to these, safe working procedures and techniques together with safety equipment complement a containment laboratory.

Primary containment provides the protection of personnel and the immediate laboratory environment from exposure to infectious agents and is provided by good microbiological technique and the use of appropriate safety equipment, such as BSC. Secondary containment is the protection of the environment external to the laboratory from exposure to infectious materials and is provided by a combination of facility design and operational practices.

1.4 Specific Containment Principles

At the lowest level of biocontainment, the containment zone may only be a chemical fume hood. At the highest level, the containment involves isolation of the organism by means of building systems, sealed rooms, sealed containers, personal protective equipment, and detailed procedures for entering the laboratories, coupled with decontamination procedures when leaving them. In most cases, this also includes high levels of security for access to the facility, ensuring that only authorized personnel may be admitted to such laboratories.

The following list describes the different specific measures of the BSLs 1–4 laboratories.

Acknowledgement

Preface

List of Contributors

Introduction

References

Chapter 1

Laboratory Biosafety in Containment Laboratories

1.1 Routes of Infection

1.2 Classification of Microorganisms

1.3 General Containment Principles

1.4 Specific Containment Principles

1.4.1 Biosafety Level 1 Laboratory