Biological agents usually have a certain route of entry into the body, although in some cases, infection can occur via multiple routes. Bacillus anthracis (the causative agent of anthrax), for example, is infectious via inhalation, percutaneous entry, or ingestion. It is important to note that natural routes of transmission and pathogenicity can differ from those associated with laboratory-acquired infection. This could be due to the use of high concentrations and high volumes of fluids, the generation of splashes and aerosols, use of sharps, and working with infected animals. In addition, the disease may also present itself differently if there has been an unconventional route of transmission. The nature of microorganisms being handled in the laboratory may vary, but one of the determining factors for an increased likelihood of microbiological exposure is when they are present at high titers as a result of their intensive propagation.

Using the inadvertent spillage of a bacterial culture as an example, the numbers of microorganisms involved will depend on the natural growth rate and age of the culture under laboratory growth conditions. A bacterial culture in late exponential growth typically has between 10⁶ and 10⁹ colony-forming units per millilitre (or milliliter) (CFUs)/mL,⁸ whereas cell concentration techniques may increase this to between 10¹⁰ and 10¹¹ CFUs/mL. Viral particles can be similarly propagated and concentrated, for example, by filtration and centrifugation, to achieve high viral levels, usually measured as plaque-forming units per millilitre (or milliliter) (PFUs/mL) or ID₅₀. Although less commonly used, fungal cultures for pathogen testing (eg, Coccidioides immitis⁹) produce stock suspensions of between 10⁷ and 10⁸ spores/mL. Therefore, there is the potential for significant exposure to microorganisms even when small volumes of cultures are being handled.

The main routes of infection in the laboratory are discussed in the following sections.

Within the laboratory, routes of percutaneous transmission can include

Blood-borne viruses (BBVs) are transmitted in contaminated blood or other body fluids following entry into the body of a susceptible person. The rate of viral transmission is dependent on the level of exposure to the virus, the type of virus, and the immune status of the exposed person. The BBVs are generally not thought to be transmitted via the respiratory route, although this possibility cannot be dismissed entirely if, under laboratory conditions such as in high-titer in vitro cultures, BBVs are present in concentrations far exceeding that found in normal body fluids. In the laboratory setting, BBVs are mainly transmitted by direct exposure to infected blood or other body fluids contaminated with infected blood, most likely via sharps injury.

Because most microbiology laboratory work involves handling cultures in aqueous suspension, splashing onto the mucous membranes is a real and feasible route of exposure and potential transmission through laboratory incidents, resulting from loss of primary containment. Aerosols may be created by, for example, spills or dropped and broken flasks.¹⁰ The loss of primary containment in these situations is likely to be in the form of material derived from a splash (see in the following text) and subsequently transferred on to other surfaces by the hands of workers.¹¹,¹²,¹³

Damage to the skin barrier such as cuts, abrasions, or lesions caused by ongoing dermatological conditions can leave a person generally more susceptible to percutaneous transmission of infection. Consequently, it is important that laboratory workers are aware of any skin conditions, including broken or punctured skin and allergies, prior to handling biological agents or potentially contaminated materials, and take any additional precautions necessary to avoid exposure.

Direct contact with microbiologically contaminated objects or surfaces (fomites) can readily result in

The transfer of microorganisms, or other hazardous substances, via surfaces and objects is a well-known exposure route and has been particularly well studied in the health care settings. In poorly controlled work environments, there is a risk of cross-contamination from hazardous materials on surfaces and inadvertent transfer, for example, via gloved or unprotected hands.¹² Human factors are also a significant factor. Even in a laboratory setting where the risks of hand to face contact are stressed, those contacts may still be made. In a study of 93 laboratory workers at BSL-2, 67 (72%) touched their face at least once, with contact to the nose being most common (44.9% of contacts), followed by contact with the forehead (36.9%), cheek/chin (12.5%), mouth (4.0%), and eye (1.7%).¹⁴ Another potential contact exposure scenario is while cleaning up a spill, warranting additional protection. The prevention of worker exposure via contact is therefore dependent on the effective use of primary containment, good laboratory technique and, where required, the decontamination of affected surfaces and equipment. Any intervention in the laboratory setting is also likely to include a disinfection step. The diligence of the operator and appropriateness of any products used for cleaning and disinfection are therefore critical to effective worker protection from surface contamination.

The airborne route of exposure is commonly implicated in occupational exposures to all types of hazards including chemicals, nonbiological particulates, and bioaerosols. It is also an exposure that can be readily controlled in the laboratory setting using good laboratory techniques and effective engineering solutions, especially ventilation controls. Failure to do this can result in inhalation of infectious aerosols, conjunctival exposure, inhalation of infectious aerosols followed by swallowing/ingestion (eg, with norovirus), and droplet exposure.

Pottage et al¹¹ examined the potential for routine microbiological techniques, such as serial dilution and pipetting, to be transmission routes by the generation of bioaerosols or splashing. Test activities, undertaken by staff with a range of experience and training, showed that aerosol and splash contamination was produced by all users, even those highly trained and experienced but that a correctly operating biological safety cabinet (BSC) will contain these aerosols. More experienced operators did not necessarily generate fewer aerosols, but those trained to work at BSL-3 did, suggesting that good microbiological technique was important. When preparing serial dilutions, those not trained at BSL-3 generated significantly more aerosols on average by over seven times. The study also showed that splash contamination on gloves and surfaces could potentially be spread within and outside the laboratory, emphasizing the importance
of effective personal protective equipment (PPE) and hygiene controls.

Working with animals, both large and small, may involve handling pathogenic microorganisms, and the animal’s physical activity may be the source of infectious bioaerosols. The containment of contaminated materials, such as fecal or urine-contaminated bedding, may be challenging under such circumstances. One study used a mouse parvovirus (MPV) model to assess the potential transfer from rodent housing containment to surrounding surfaces.¹⁵ The chosen virus represented the possible cross-contamination routes for other harmful viruses. The study found little evidence of significant deposition of the virus onto transfer surfaces, such as gloves and work surfaces outside of contained rodent storage cages. Even the positive pressure in ventilated cages did not result in animal room floor contamination, a suspected bioaerosol route of transfer, despite exhaust air escaping from the cage-top lip prior to high-efficiency particulate air (HEPA) filtration. However, soiled animal bedding and its handling by laboratory personnel was implicated in bioaerosol generation.

The ability of a pathogen to cause infection requires its contact with susceptible host receptors (a combination of routes of transmission and exposure, as mentioned earlier) in a sufficient quantity, that is, at an infectious dose. Data on infectious dose for the wide range of human pathogens are limited. However, some notable examples illustrate the importance of taking this into consideration. Escherichia coli is an ubiquitous bacterium. Most E coli strains can cause minor infection via the oral route (hand-to-mouth transfer) but require a high dose to do so; therefore, it is safe to work with them in a general-purpose microbiology laboratory (BSL-2) using routine laboratory precautions. Disabled strains are commonly used tools in laboratory work, especially in genetic and molecular biology modifications and because of this attenuation, they are of minimal hazard (equivalent of BSL-1). However, verocytotoxigenic strains of E coli, such as O157, are zoonotic agents that can cause severe infection in humans at a very low infectious dose, estimated at just tens or hundreds of cells.¹⁶,¹⁷ This E coli serotype therefore requires laboratory handling using enhanced precautions (equivalent to BSL-3) that minimize exposure.

Where data on infectious dose are available, it is important to factor this into the biohazard assessment in developing a risk assessment. In addition to evidence from research papers, public and occupational health Web sites also provide a valuable source of data on infectious dose of human pathogens (eg, European Centre for Disease Prevention and Control,¹⁸ World Organisation for Animal Health,¹⁹ Public Health Agency of Canada,²⁰ Health and Safety Executive,²¹ Public Health England,²² Centers for Disease Control and Prevention,²³ World Health Organization²⁴). However, such data must be used with caution because it is typically based on infectious dose derived from natural routes of transmission. In the laboratory, much higher titers of pathogens may be handled in culture and laboratory activities and, as described in the following text, this may create different potential routes of transmission. For example, although some vector-borne pathogens are most likely to be transmitted in the laboratory when handling infected mosquitoes or ticks, percutaneous injury from contaminated sharps may mimic the vector-borne route. These laboratory-specific routes of transmission must be taken into consideration.

Environmental robustness, that is, persistence of a pathogen outside its host, or outside of ideal culture conditions, is a further factor in assessing biohazard. At the one extreme, some enveloped viruses are more sensitive to ambient humidity and pH and quickly lose viability on surfaces or in aerosols,²⁵ whereas at the other extreme, spore-forming bacteria have been shown to survive in the wider environment for decades.²⁶