Avoiding biohazards in medical, veterinary and research laboratories

Personnel in medical, veterinary or research laboratories may be exposed to a wide variety of pathogens that range from deadly to debilitating. For some of these pathogens, no treatment is available, and in other cases the treatment does not fully control the disease. It is important that personnel in laboratories that process human or microbiological specimens follow universal precautions when handling tissues, cells, or microbiological specimens owing to the increasing numbers of individuals infected with hepatitis C and HIV in the US and the possibility that an individual may be asymptomatic when a specimen is obtained. Similar precautions must be followed in laboratories that use animal tissues owing to the possibility of exposure to agents that are pathogenic in humans. Personnel with conditions associated with immunosuppression should evaluate carefully whether or not specific laboratory environments put them at increased risk of disease. We offer here some general approaches to identifying biohazards and to minimizing the potential risk of exposure. The issues discussed can be used to develop a general safety program as required by regulatory or accrediting agencies, including the Occupational Safety and Health Administration.     

SUICIDE PROBLEMS IN MEDICAL PRACTICE

Increasing awareness by physicians in general medical practice of the possibility of suicide in nonpsychiatric patients is indispensable for the evaluation of suicide risk and for a practical approach to the problem of prevention.An analysis was made of the records of 11 cases of suicide by medical and surgical patients who were in a general nonpsychiatric Veterans Administration hospital for evaluation and treatment of physical disease.It was noted that the general hospital staff had a low index of suspicion of the possibility of suicide in general hospital patients.It appeared from this study that there is a definite suicide risk in older persons hospitalized for physical illness who develop psychotic reactions during the course of their illness and hospitalization. The signs and symptoms of toxic and organic psychosis in these older patients were not recognized and their significance relative to suicide risk was not appreciated. Although psychiatric signs of severe emotional disturbance appeared clinically obvious at least 24 hours before suicide in ten of the eleven patients, no suicide risk was considered present, nor were adequate precautions taken by the hospital staff. This was owing to the lack of psychiatric orientation among the nonpsychiatric physicians.     

Diagnosis of active tuberculosis in a university hospital

Active tuberculosis was diagnosed in 100 patients at Sunnybrook Medical Centre during the five-year period 1968-72. These cases were studied to find out if any delay had taken place in establishing the diagnosis and starting treatment. Delay was found most frequently when patients presented with atypical disease or when microbiological investigations were negative or misinterpreted. However, in nine patients with positive Ziehl-Neelsen smears the diagnosis was delayed more than one week. Usually such delays were caused by a failure to send specimens promptly for examination for acid-fast bacilli. Lack of communication between the laboratory and the clinician was found to be responsible for delays in starting treatment in several patients. It is essential that the clinician in a general hospital be alert to the possibility of tuberculosis and that there be close cooperation between the clinical staff and the diagnostic services.     

Alternative, cost-effective fungus-staining method for control slides in cytology and histopathology

OBJECTIVE: To develop a cost-effective, reliable and safe method of providing fungal control slides for routine use in pathology laboratories. STUDY DESIGN: A set of easily available, low-cost material was tested to obtain fungal colonies on substrate adequate for paraffin-embedded sections or smears. RESULTS: Such material as cheese is a simple, inexpensive and practical culture medium for silver-positive fungi. A batch of paraffin blocks can be prepared to maintain a stock of control material in the laboratory. CONCLUSION: It is useful to maintain fungal colonies to produce staining control specimens using small pieces of refrigerated cheese to easily produce silver-staining control specimens or smears embedded in paraffin, reducing the risk of accidental exposure to potentially infective pathogens in the laboratory. This method might also be a good alternative for conserving routine surgical specimens, considering the currently decreasing numbers of necropsy and large specimens, particularly from immunosuppressed and infected patients.     

Hydrogen peroxide vapour (HPV) inactivation of adenovirus

Aims: Adenovirus contamination can be problematic in various settings including life science laboratories and during pharmaceutical manufacturing processes. Stringent and effective decontamination procedures are necessary to minimize the risk of personnel exposure or product cross‐contamination in these settings. Hydrogen peroxide vapour (HPV) is sporicidal, tuberculocidal and fungicidal with proven efficacy against some viruses. We investigate the efficacy of HPV for the inactivation of a recombinant adenovirus. Methods and Results: In this study, the survival of a dried recombinant adenovirus (Ad5GFP) was tested before and after HPV exposure to determine the efficacy of HPV at inactivating adenovirus. A > 8‐log TCID₅₀ reduction resulted from 45‐min exposure to HPV in a microbiological safety cabinet. Conclusions: HPV is effective for the inactivation of a recombinant adenovirus. Significance and impact of the study: The results suggest that HPV may be useful for adenovirus decontamination in life science laboratories or in manufacturing facilities.     

A risk assessment approach for fresh fruits

Aims:  To describe the approach used in conducting a fit‐for‐purpose risk assessment of microbiological human pathogens associated with fresh fruit and the risk management recommendations made. Methods and Results:  A qualitative risk assessment for microbiological hazards in fresh fruit was carried out based on the Codex Alimentarius (Codex) framework, modified to consider multiple hazards and all fresh (whole) fruits. The assessment determines 14 significant bacterial, viral, protozoal and nematodal hazards associated with fresh produce, assesses the probable level of exposure from fresh fruit, concludes on the risk from each hazard, and considers and recommends risk management actions. A review of potential risk management options allowed the comparison of effectiveness with the potential exposure to each hazard. Conclusions:  Washing to a recommended protocol is an appropriate risk management action for the vast majority of consumption events, particularly when good agricultural and hygienic practices are followed and with the addition of refrigerated storage for low acid fruit. Additional safeguards are recommended for aggregate fruits with respect to the risk from protozoa. Significance and Impact of the Study:  The potentially complex process of assessing the risks of multiple hazards in multiple but similar commodities can be simplified in a qualitative assessment approach that employs the Codex methodology.     

Microbial hazards in plant tissue and cell cultures

Summary A wide range of microorganisms (filamentous fungi, yeasts, bacteria, viruses and viroids) and micro-arthropods (mites and thrips) have been identified as contaminants in plant tissue cultures. Contaminant may be introduced with the explant, during manipulations in the laboratory or by micro-arthropod vectors. Contaminants may express themselves immediately or can remain latent for long periods of time. This often makes it difficult to identify the source of contamination. Disinfection protocols have now been developed for a wide range of plant species including those infected with viruses/viroids or endophytic bacteria. They may include the selection of pathogen-free donor plants or donor plant treatments such as thermotherapy. Also microbiological quality assurance systems (e.g. Hazard Analysis Critical Control Point; HACCP procedures) have been adapted to the needs of commercial plant tissue culture laboratories. These are aimed at, preventing the introduction of pathogens, into tissue cultures at establishment and in the laboratory. In established in vitro cultures preventative strategies have proved to be essential, since it is extremely difficult to eliminate environmental bacterial and fungal contaminants using, antibiotics and fungicides. In many cases anti-microbial treatments only inhibit contaminants and low levels of contamination persist. In particular, the use of antibiotics against Gram-negative bacteria (including plant pathogenic bacteria and Agrobacterium tumefaciens vector systems used in genetic engineering) has been shown frequently to be extremely difficult or unsuccessful. Detection of latent contamination may involve the use of general and semi-selective microbial growth media or serological and PCR-based molecular techniques for specific pathogens. However, it is often difficult to detect low numbers of latent bacterial contaminants (e.g. levels present following antibiotic treatment or when acidified plant media are used). This poses a particular risk in the production of transgenic plants where the elimination or detection of Agrobacterium tumefaciens-based vector systems cannot be guaranteed with the currently available methodologies. Recent research has also shown that there is a risk of the transmission of human pathogens in plant tissue cultures.     

Pathogen and rodenticide exposure in American badgers (Taxidea taxus) in California

Urban and agricultural land use may increase the risk of disease transmission among wildlife, domestic animals, and humans as we share ever-shrinking and fragmented habitat. American badgers (Taxidae taxus), a species of special concern in California, USA, live in proximity to urban development and often share habitat with livestock and small peridomestic mammals. As such, they may be susceptible to pathogens commonly transmitted at this interface and to anticoagulant rodenticides used to control nuisance wildlife on agricultural lands. We evaluated free-ranging badgers in California for exposure to pathogens and anticoagulant rodenticides that pose a risk to wildlife, domestic animals, or public health. We found serologic evidence of badger exposure to Francisella tularensis, Toxoplasma gondii, Anaplasma phagocytophilum, canine distemper virus, and three Bartonella species: B. henselae, B. clarridgeiae, and B. vinsonii subsp. berkhoffii. Badger tissues contained anticoagulant rodenticides brodifacoum and bromadiolone, commonly used to control periurban rodent pests. These data provide a preliminary investigation of pathogen and toxicant exposure in the wild badger population.     

Identification of bacterial strains by laboratories participating in the Deutsches Krebsforschungszentrum quality assurance programme

The quality assurance programme (QAP) of the Deutsches Krebsforschungszentrum (DKFZ) is a proficiency testing system developed to service the laboratory animal discipline. QAP comprises the quarterly distribution of two bacterial strains originating from various species of animals for identification to the species level and antibiotic susceptibility testing. We compared identification results reported by QAP participants over the years 1996-2004 with those obtained by the Dutch Bacterial Diagnostics reference laboratory on 68 samples comprising 71 bacterial strains and a fungus. Significant differences were found in the frequency of reported and correct identifications when bacteria were assigned to different groups based on morphology by Gram stain and on origin (animal versus environmental, rodent and rabbit versus other animal species, pathogen versus non-pathogens). Rodent and rabbit pathogens yielded 73% correct identifications, and with all bacterial strains only 60% of the identifications were correct. We assume that most QAP participants were from laboratory animal diagnostic laboratories. If this is true, the capabilities of laboratories in the laboratory animal discipline to correctly identify bacterial species are well below what are considered acceptable limits for human diagnostic laboratories. The distribution of cultured bacteria circumvents the most difficult step in the microbiological monitoring of animals, namely primary culture from clinical samples. We propose to set up a QAP that comprises the distribution of specimens mimicking clinical samples normally submitted to laboratory animal diagnostic laboratories.     

Cluster of three cases of invasive meningococcal disease in a preschool facility in West Bohemia, the Czech Republic

From 1993 to 2009, there was only one cluster of invasive meningococcal disease (IMD) reported in a community of children in the Czech Republic. This exceptional cluster that occurred in a preschool facility is the focus of this report. In response to the announcement of the disease, anti-epidemic precautions were put in place. Neisseria meningitidis isolates were delivered from local laboratories to the National Reference Laboratory for Meningococcal Infections in Prague. Phenotyping was performed there along with multilocus sequence typing. Related factors and microbiological results were analyzed retrospectively. In October 2009, three girls contracted IMD within a period of 1 week in a 42-member group in a preschool facility attached to the elementary school in Starý Plzenec-Sedlec. In relation to three cases of the disease, another 66 people were registered of which 58 underwent a microbiological examination. N. meningitidis was detected in a total of five (8.6 %) people. The National Reference Laboratory for Meningococcal Infections defined the type of the strain to be C: P1.18-1,34-2,38: F1-7: ST-467 (cc269) and penA27. Tests showed the precise identity of all strains obtained from the three sick children and of two strains contracted through contact with the preschool facility. Despite the complete recovery of all patients with no permanent damage, the need for rapid cooperation between clinical sites, diagnostic laboratories, and epidemiologists was confirmed.     

Acquired immunodeficiency syndrome. Measures to be taken in blood transfusion establishments in order to protect the personnel

There is presently no evidence of AIDS transmission to laboratory workers. Because transmissible agent is suspected, precautions should be taken in laboratories. As a general rule, procedures appropriate for hepatitis B prevention are advised. These procedures concern blood collection, carrying, and processing. Measures should be taken in order to avoid blood spilling and work surfaces should be decontaminated. Disposable material should be preferred, and all potentially contaminated material should be decontaminated before disposal or reprocessing. All personnel should be aware of usual laboratory procedures concerning pipetting, hand washing, protective clothing and of general precautions against viral contaminations.     

Microbial risk assessment: don't forget the children

Quantitative microbial risk assessment is a rapidly developing field with a purpose to quantify risks of infection, disease and mortality from the environmental exposure of pathogens. It is currently being applied to the development of standards for drinking water, wastewater re-use and foods. A growing body of evidence indicates that the greatest risk of infection for enteric pathogens is for persons less than 19 years of age. Children are more likely to become ill from consumption of contaminated drinking water and recreational activities. These increased risks may be because immunological, neurological and digestive systems are still developing. In addition, children are more environmentally exposed to pathogens. For some enteric pathogens children may be the greatest at risk population.     

Stress and microbial endocrinology: prospects for ruminant nutrition

The feed efficiency of ruminant meat and dairy livestock can be significantly influenced by factors within their living environments. In particular, events perceived by the animals as stressful (such as parturition, transport or handling) have been found to affect susceptibility to infection. It has been well documented that even minor stress such as weighing can result in an increase in colonisation and faecal shedding of enteric pathogens such as Salmonella enterica and Escherichia coli O157:H7. Such infections affect both ruminant overall health and therefore performance, and are a particular problem for the meat production industries. Prior explanations for stress enhancing the likelihood of infection is that activation of the sympathetic nervous system under stress leads to the release of neuroendocrine mediators such as the catecholamine stress hormones noradrenaline and adrenaline, which may impair innate and adaptive immunity. More recently, however, another equally compelling explanation, viewed through the lens of the newly recognised microbiological discipline of microbial endocrinology is that the myriad of bacteria within the ruminant digestive tract are as responsive to the hormonal output of stress as the cells of their host. Work from our laboratories has shown that enteric pathogens have evolved systems for directly sensing stress hormones. We have demonstrated that even brief exposure of enteric pathogens to physiological concentrations of stress hormones can result in massive increases in growth and marked changes in expression of virulence factors such as adhesins and toxins. Happy, less stressed ruminants may therefore be better-nourished animals and safer sources of meat. This article reviews evidence that stress, as well as affecting nutrition, in ruminants is correlated with increased risk of enteric bacterial infections, and examines the molecular mechanisms that may be at work in both processes.     

Contribution of the hydrophobic effect to microbial infection

The hydrophobic effect has been known for decades. Numerous researchers have invoked the hydrophobic effect to explain how pathogens adhere to tissues. In some cases, inhibition of adhesion can be brought about by low concentrations of aromatic compounds, such as p-nitrophenol or tryptophan. Because the hydrophobic effect has been considered to be nonspecific, the molecular biology of adhesive hydrophobins has not been studied in as much detail as lectin adhesins. The literature provides compelling evidence that a large number of bacterial and fungal pathogens depend on hydrophobic interactions for successful colonization of a host. Several laboratories are now developing effective antiadhesins, based on inhibition of hydrophobic interactions between the host and the pathogen.     

Polymicrobial infections in children with diarrhoea in a rural area of Jordan

Polymicrobial infections associated with diarrhoea are common in developing countries. Stool specimens were collected from 220 patient children and 100 controls. Potential pathogenic agents isolated from 143 (65%) children were identified by molecular and standard microbiological methods. Co-infections with two or more agents were detected in 50 (35%) cases. Escherichia coli, Shigella dysenteriae, Giardia and Entamoeba histolytica were found to be predominant. The etiologic agents could not be determined in 77 (35%) cases. The most significant risk factors were the age, the education level of the mother and the use of non-chlorinated water. The high infection rate of diarrhoeal diseases is a strong indication that these pathogens circulate easily through the population.     

Prioritizing risks and uncertainties from intentional release of selected Category A pathogens

This paper synthesizes available information on five Category A pathogens (Bacillus anthracis, Yersinia pestis, Francisella tularensis, Variola major and Lassa) to develop quantitative guidelines for how environmental pathogen concentrations may be related to human health risk in an indoor environment. An integrated model of environmental transport and human health exposure to biological pathogens is constructed which 1) includes the effects of environmental attenuation, 2) considers fomite contact exposure as well as inhalational exposure, and 3) includes an uncertainty analysis to identify key input uncertainties, which may inform future research directions. The findings provide a framework for developing the many different environmental standards that are needed for making risk-informed response decisions, such as when prophylactic antibiotics should be distributed, and whether or not a contaminated area should be cleaned up. The approach is based on the assumption of uniform mixing in environmental compartments and is thus applicable to areas sufficiently removed in time and space from the initial release that mixing has produced relatively uniform concentrations. Results indicate that when pathogens are released into the air, risk from inhalation is the main component of the overall risk, while risk from ingestion (dermal contact for B. anthracis) is the main component of the overall risk when pathogens are present on surfaces. Concentrations sampled from untracked floor, walls and the filter of heating ventilation and air conditioning (HVAC) system are proposed as indicators of previous exposure risk, while samples taken from touched surfaces are proposed as indicators of future risk if the building is reoccupied. A Monte Carlo uncertainty analysis is conducted and input-output correlations used to identify important parameter uncertainties. An approach is proposed for integrating these quantitative assessments of parameter uncertainty with broader, qualitative considerations to identify future research priorities.     

Studying fungal pathogens of humans and fungal infections: fungal diversity and diversity of approaches

Seminal work by Louis Pasteur revealed the contribution of fungi – yeasts and microsporidia to agroindustry and disease in animals, respectively. More than 150 years later, the impact of fungi on human health and beyond is an ever-increasing issue, although often underestimated. Recent studies estimate that fungal infections, especially those caused by Candida, Cryptococcus and Aspergillus species, kill more than one million people annually. Indeed, these neglected infections are in general very difficult to cure and the associated mortality remains very high even when antifungal treatments exist. The development of new antifungals and diagnostic tools that are both necessary to fight fungal diseases efficiently, requires greater insights in the biology of the fungal pathogens of humans in the context of the infection, on their epidemiology, and on their role in the human mycobiota. We also need a better understanding of the host immune responses to fungal pathogens as well as the genetic basis for the increased sensitivity of some individuals to fungal infections. Here, we highlight some recent progress made in these different areas of research, in particular based on work conducted in our own laboratories. These progresses should lay the ground for better management of fungal infections, as they provide opportunities for better diagnostic, vaccination, the development of classical antifungals but also strategies for targeting virulence factors or the host.     

Molecular characterization of ticks and tick-borne pathogens

Ticks and their vertebrate hosts often carry several pathogens simultaneously, which either belong to different or to the same genera. Conventional methods (such as blood smear examination or tick salivary gland staining) are often unable to discriminate between pathogens. Therefore, molecular methods for the detection and differentiation of tick-borne pathogens are increasingly used. Technical problems still remain to identify pathogens within tick tissues, or within host animals when infection rates are very low. Recently we developed an integrated approach to identify several pathogens with only one molecular test. This approach, the reverse line blot hybridization (RLB) reduces costs of analysis, gives quicker results. and allows standardized inter-laboratory comparisons. Finally, this paper also focuses on the molecular diagnostic techniques currently used in the laboratories of the Mediterranean countries.     

Survival curves for normal-tissue clonogens: a comparison of assessments using in vitro, transplantation, or in situ techniques

A survey of survival curves in the literature, for clonogenic cells (clonogens) in normal tissues, highlights the following features: the sensitivity of some human and dog clonogens apparently is greater than that of their counterparts in mice and sheep, assessed in vitro. However, this should be interpreted with caution because of the possibility of cell selection and the ability to modify sensitivity markedly in some systems by variations in growth conditions; extrapolation numbers are in general higher when assessed in vivo than in vitro. This is due partly to the lack of measurements of repair of potentially-lethal damage using many assays in vitro. This feature increases the extrapolation number when measured using transplantation assays in vivo; epithelial clonogens in vivo demonstrate a remarkable similarity in sensitivity between tissues. The range is similar for clonogens assayed in situ or by transplantation, and this argues against the possibility that a resistant subpopulation may be selected in most assays in situ. It is emphasized from the comparisons that caution must be exercised in extrapolating results, obtained for clonogens assayed in vitro or by transplantation in vivo, to the situation in situ.     

Quantitative risk assessment of Listeria monocytogenes in ready-to-eat foods: the FAO/WHO approach

Quantitative microbiological risk assessment is a very new and unique scientific approach able to link, for the first time, data from food (in the farm-to-fork continuum) and the various data on human disease to provide a clear estimation of the impact of contaminated food on human public health. The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) have recently launched risk assessment studies of a number of pathogen-food commodity combinations (Salmonella in eggs and in broiler chickens, Listeria monocytogenes in ready-to-eat foods, Campylobacter in broiler chickens, Vibrio in seafood) to be used to lower the risk associated with these food-borne diseases and ensure fair practices in the international trade of food. The FAO/WHO Listeria risk assessment was undertaken in part to determine how previously developed risk assessments done at the national level could be adapted or expanded to address concerns related to L. monocytogenes in ready-to-eat foods at an international level. In addition, after initiation of the risk assessment, the risk assessors were asked by the Codex Committee on Food to consider three specific questions related to ready-to-eat foods in general, which are: (1). estimate the risk for consumers in different susceptible populations groups (elderly, infants, pregnant women and immunocompromised patients) relative to the general population; (2). estimate the risk for L. monocytogenes in foods that support growth and foods that do not support growth under specific storage and shelf-life conditions; (3). estimate the risk from L. monocytogenes in food when the number of organisms ranges from absence in 25 g to 1000 colonies forming units per gram or milliliter, or does not exceed specified levels at the point of consumption. To achieve these goals, new dose-response relationships and exposure assessments for ready-to-eat foods were developed. Preliminary data indicate that eliminating the higher dose levels at the time of consumption has a large impact on the number of predicted cases.