IN CLINICAL
JULY
MICROBIOLOGY
1996
Laboratory Safety i? Clinical Microbiology R. JAMISON, MICHAEL and JOHN A. SMITH
COORDINATING
A. NOBLE,
EILEEN
EDITOR
JOHN A. SMITH
AMERICAN
SOCIETY
FOR MICROBIOLOGY
M. PROCTOR,
Cumitcch IA l Blood Cultures II l June 1982 Cumitech 2A l Laboratory Diagnosis of Urinary Tract Infections l March 1987 Cumitech 3A l Quality Control and Quality Assurance Practices in Clinical Microbiology l May 1990 Cumitcch 4A l Laboratory Diagnosis of Gonorrhea l April I993 Cumitech SA l Practical Anaerobic Bacteriology l December 1991 Cumitcch bA l New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide l February 1Y9 1 Cumitcch 7A l Laboratory Diagnosis of Lower Respiratory Tract Infections l September 1087 Cumltcch 8 l Detection of Microbial Antigens by Countcrimmunoclectrophoresis l Deccmbcr 197X Cumitcch 9 l Collection and Processing ot Bacteriological Specimens l August 197Y Cumitech 10 l Laboratory Diagnosis of Upper Respiratory Tract Infections l December 1979 Cumitech 1 I l Practical Methods for Culture and Identification of Fungi in the Clinical Microbiology Laboratory l August 1980 Cumitcch 12A l Laboratory Diagnosis of Bacterial Diarrhea l April 1992 Cumitcch 13A l Laboratory Diagnosis of Ocular Infections l Septcmbcr 1994 Cumitech 14A l Laboratory Diagnosis of Central Nervous System Infections l March 1YY3 Cumitech 1SA l Laboratory Diagnosis of Viral Infections l August 1994 Cumitcch I6A l Laboratory Diagnosis of the Mycobactcrioses l October 1994 Cumitcch 17A l Laboratory Diagnosis of Female Genital Tract Infections l June 1993 Cumitcch 18 l Laboratory Diagnosi\ of Hepatitis Viruses l January 1984 Cumitcch 1Y l Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections l August 1084 Cumitech 20 l Therapeutic Drug Monitoring: Antimicrobial Agents l October 1984 Cumitcch 21 l Laboratory Diagnosis of Vn-al Respiratory Disease l March 1986 Cumitcch 22 l Immunoscrology of Staphylococcal Disease l August 1987 Cumitcch 23 l Infections of the Skin and Subcutaneous Tissues l June 1988 Cumitech 24 l Rapid Detection of Viruses by ImmunofIuorescence l August I988 Cumitcch 25 l Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing l Dcccmbcr 1988 Cumitech 26 l Laboratory Diagnosis of Viral Infections Producing Enteritis l Scptcmber 1989 Cumitech 27 l Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Companion and Laboratory Animals l February 1996 Cumitech 28 l Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from Companion and Laboratory Animals l February 1996 Cumitech 29 l Laboratory Safety in Clinical Microbiology l July 1996 Cum/tech Cumltech MicrobIology,
29,
should Laboratory Washington,
be cited Safety D C
as follows, In Cllnlcal
Editorial Board for ASM Cumitechs: Gleaves,
Janet
Hindler,
Thomas
J. Inzana,
e g
Jamtson,
R , M A
MIcrobiology.
Frederick Brenda
S. Nolte, McCurdy,
Noble,
Coordlnatlng
Chairman; John
A. Smith,
E M
Proctor,
ed , J A.
Lorraine Alice
Smith
Clarke, S. Weissfeld,
and
J A
Smith.
American
Mary
1996
Society
J. R. Gilchrist, and
Stephen
Curt Young.
The purpose of the Cumltech series IS to provide consensus recommendations by the authors as to appropriate state-of-the operating procedures for clerical microbiology laboratones which may lack the facllltles for fully evaluattng routine or new methods The procedures qven are not proposed as ’ standard” methods
Copyright
0 1996 American Society for Microbiology 1325 Massachusetts Ave., N.W. Washington, DC 20005
for
art
LABORATORY R JAWSON, 3932
Department
SAFETY IN CLINICAL
of Pediatrics,
Louisiana
MICHAEL British
A. NOBLE, Division of Medical Columbia, Canada V5Z lM9
EILEEN IL8
M. PROCTOR,
JOHN A. SMITH, British Columbia,
British
Columbia
Division of Medical Vancouver, British
State University
Microbiology,
Division of Medical Vancouver, British
GENERAL
Center,
Hospital
Control,
Microbiology, Vancouver Hospital Columbia, Canada V5Z lM9 COORDINATING
JOHN A. SMITH, British Columbia,
Medical
Vancouver
Centre for Disease
MICROBIOLOGY Shreveport,
& Health
Vancouver,
Louisiana
Sciences
British
Centre,
Columbia,
71130-
Vancouver,
Canada
V5Z
& Health
Sciences
Centre
and University
of
& Health
Sciences
Centre
and University
of
EDITOR
Microbiology, Vancouver Hospital Columbia, Canada V5Z lM9
INTRODUCTION
Laboratory supervisorsare responsiblefor providing all personsunder their supervisionwith notification of potential hazardsand for training personnelin safelaboratory practices.In addition, supervisorsmust correct work errors and conditions that may result in personalinjury, including accidentalexposureto infectious agents;provide for adequate safety equipment (containers, eye washstations); and help develop a positive attitude toward safety in the laboratory. The laboratory shouldhave a safety officer who is responsiblefor ensuringthat there is a safety manualthat includesall of the laboratory’s safety rules and that all employeesread it and sign and date a sheetindicatingthat they have read it. This manualshouldbe revisedat leastonce annuallyas well aswhen new material is added, and employees should be reorientated annually. The safety manualshouldbe customdesignedfor the particular laboratory and should comprehensivelyand explicitly cover all the hazards associatedwith microbiologicaltesting,including thoseassociated with specimencollection, transportation, accession, and processingand safe disposalof wastes, both in institutional and in community waste disposalsites. The manual also should address physical,chemical,environmental,and ergonomic threatsto the health of workersand others.There shouldbe referencesto legislationand guidelines concerningsafety from various federal, state, and local agencies,including the Occupational Safety and Health Administration (OSHA), the National Institute for OccupationalSafety and Health, the National Institutes of Health, the Centers for DiseaseControl and Prevention (CDC), the International Air TransportationAuthority, and other legislativeauthorities. 1
Safety goals can be met by providing written proceduresand adequateequipmentfor handling of materialscontainingpotentiallyinfectiousagents. For instance,containersfor disposalof contaminated pipettes,beakers,and tubesmustbe readily available and sufficiently portable to allow for later autoclaving.Properly labelledcontainersfor contaminated wastes must be placed in close proximity to the work area for temporary storage of the waste prior to either incineration or autoclaving. Pipette jars containing an adequatedisinfectant and sharpscontainers must be conveniently placedin the work area.Containmentareas suitablefor the task at hand, including biological safety cabinetsappropriatefor the pathogen,flammablesstorage and storage cabinets, and fume hoodsfor handling volatile or noxious materials, must be provided. All laboratory workers are responsiblefor complying with safety rules, regulations, and proceduresrequired for the task assigned,for their own protection aswell asthat of their fellow workers and the public. It is expected that the laboratory workers will cooperate by keeping their immunization historiescurrent or providing written documentation that they choosenot to be vaccinated againstspecific infectious agents (e.g., influenza virusesand hepatitis B virus). They are responsible for reporting all facts pertaining to every incident with the potential for personalinjury or exposure to infectious agents,and any situation that could result in such an incident, to their immediate supervisor. Each laboratory worker must use personal protective equipment (e.g., gowns,gloves, and boots) when appropriate and comply with regulations governing frequency of changeand methodsof disposalof the clothing, as well as regulationsregardingwear outside of the
2
JAMISON ET AL.
CUMITECH
29
work area. Eating, drinking, smoking,chewingof cavalier attitudes towards safety on the part of gum or tobacco, and application of cosmeticsin workers. The relationship between microbiologithe laboratory mustbe prohibited. Food mustnot cal laboratory infections and human factors such be stored in laboratories where any infectious as attitude was studied by Phillips (36). This agentsor clinical specimensare located. Labora- case-controlstudy comparedexperiencedlaboratory workers shouldwear glovesas required and tory workers who had had laboratory accidents remove their glovesandwashtheir handsprior to within a two-year period with similarworkerswho leavingtheir work area.It is axiomatic that clinical had been accident free during the sameperiod. specimens,contaminatedmaterials,pipettes, etc., No differencesin age, education,years of experimustbe disposedof in an appropriate manner.All ence, marital status,number of children, awarepipetting must be performed with the aid of nessof safety principles, hobbies, and interests mechanicaldevices;mouth pipetting is prohibited were noted between the “accident involved” (AI) in the clinical laboratory. Laboratory workers versusthe “accident free” (AF) groups,with the must disinfect their laboratory work spacedaily singleexception that those in the AI group were and immediately after a spill or other type of more likely to smoke. Importantly, when asked contamination. about their accidentand infection recordsprior to Some laboratories take a serum samplefrom the two-year study period, individuals in the AI employeeson recruitment for comparisonwith a group revealed that they previously had experisubsequentsamplein caseof illness. enced significantly more injuries and infections Despite deficiencies,publishedstudiesprovide and non-lost-time accidents than those in the insight into the nature of laboratory-acquiredin- AF group, indicating the persistenceof accidentfections (22, 37-39).Infections causedby Brucella proneness.When questionedabout attitudes and spp. (20, 21, 32, 52), meningococcus(23), Escheequipmentuse,the AF group attachedsignificantrichia coli 0157:H7 (lo), Streptococcus spp. (40), ly greater importance to attitude towards safety, salmonellae(6,13), and HBV (19) have been doc- while the AI group rated technique as more imumented.Sourcesof infectionshavebeenassociated portant than attitude. Thirty-two of the 33 indiwith humanerror, mechanicalerror, and accidents. viduals in the AF group felt that an accident-free Punctures,cuts,abrasions,andother skinexposures work record was “something to be proud of.” are the most common accidents.Other systemic Analysis of the accidentsthe AI group had expeproblemsthat affect the microbiology laboratory riencedrevealedthat 61%were reported to be the include accidentsassociatedwith blood collecting result of an unsafeact, 15% were reported to be devices(30) and external contamination of speci- the result of equipment failure, and 21% were men containers(2). The World Health Organiza- reported to be a result of a combination of the tion has recognized that changing nomenclature two. Many of the accidentswere attributable to of pathogensmay be a hazard in clinical labora- the inability to recognize a hazardoussituation or tories if it causesconfusion or resultsin a lack of to excessiverisk taking. In another study, Blaser awarenessof the pathogensbeing handled (60). and Lofgren (9) describeda fatal Salmonella inSafety procedures in the laboratory can be fection in a family memberof an infected laboradivided into those associatedwith laboratory de- tory worker. Thus, the fundamental rule governsign,with laboratory practice, and with laboratory ing laboratory safety is to instill in the minds of waste disposal.The laboratory shouldbe located employeesthat safety is important not only to awayfrom high-traffic areasand closeto the waste employeesbut also to their colleagues,families, disposalareaif biohazardouswasteis transported. and the public at large. The laboratorymustbe ventilated accordingto preThe epidemiology of laboratory-associatedinvailing building code standardsand be equipped fections is ill defined, despitethe efforts of investo render all microbiologicalwasteproducts non- tigators who studied the subject. Several investiviable. gators have described infections in laboratory .Biosafety, a corollary of biocontamination, is workers (35, 37-39, 47, 53). However, thesestudbasedon a combination of good microbiological ies are biasedto a large extent becauseof the intechniques,goodlaboratory design,andthe useof clusionof researchlaboratories.Grist and Emslie safety equipment,both personaland environmen- (22) were able to segregateclinical laboratories tal. There are severalissuespertaining to labora- from research laboratories in describinglaboratory safety that are commonto all laboratoriesin tory infections and documenteda decreasedinciwhich infectiousagentsare handled,whether they dencein the past two decades. be clinical laboratories or those that are utilized Specimen Collection for either applied or basic microbiological research. The basic rules governing behavior of Laboratory testing begins with the collection personnelin the microbiologylaboratory are uni- and transportation of specimensto the laboratory. versal. Study results indicate that an increased Health care workers who collect specimensfrom incidenceof laboratory accidentscorrelateswith patients must exercise care in the process.All
CUMITECH
29
LABORATORY
blood is considered potentially infective. Thus, gloves must be worn during venipuncture, and phlebotomists must avoid needle stick injury. Recapping of needles is prohibited. Sputum, urine, or stool samples should be collected into a sufficiently wide-mouthed container, taking care not to contaminate the outside of the container. If this happens, the container should be discarded in favor of a repeat specimen or should be disinfected before being sent to the laboratory, depending on the ease of obtaining a repeat specimen. Requisitions should be protected from leakage of samples. These examples of worker protection indicate that the need to take precautions is not only for the sake of the specimen collector but also for the sake of the courier who transports the sample and those receiving it in the laboratory. Clinicians submitting samples to the laboratory must be informed and must understand the necessity of alerting personnel when highly pathogenic organisms are likely to be encountered (21). Specimen Packaging Specimens should be placed in an appropriate leak-proof sterile container and then placed in a secondary container, such as a clear plastic bag equipped with a separate pocket for the requisition. All specimens should be considered potentially infectious. Because of the lability of many viruses outside the body, specimens not obtained in-house may arrive frozen or refrigerated. The need to transport potentially infectious materials to the laboratory is a primary factor in the occurrence of biohazard spills. Dropping and breakage of vessels containing infectious agents are serious concerns. Whenever practical, the use of plastic containers for collection and temporary storage of clinical specimens should be encouraged. The laboratory should insist on the use of common infection control safety protocols, including the use of sealed secondary containers from bedside to the laboratory. The transport packages should be opened in an appropriate biological safety cabinet by a technologist who is properly equipped with personal protective clothing. However, biological specimens contained in a suitable transport vessel (plastic or glass) can safely be stored in the laboratory for future processing, provided that the transport vessels are assumed to be contaminated and are thus disinfected with a solution of sodium hypochlorite (100 ppm) prior to being brought out of the hood. Specimen Transportation An important criterion in selecting a specimen container is its ability to withstand the rigors of transportation, whether this be by messenger service or by mechanical device, such as a pneumatic tube. Clearly, leakage of infectious materials may constitute a health hazard to those handling the
SAFETY
IN CLINICAL
MICROBIOLOGY
3
materials that have been contaminated. Upon receipt in the laboratory, such leaking items must be wiped with disinfectant or, if easily replaced, a repeat specimen should be requested. On rare occasions, the virus laboratory may choose to furnish tubes of tissue cultures to the medical staff in order to facilitate inoculation of the clinical specimen into tissue culture at the patient’s bedside. The outside of these culture tubes should be assumed to have been contaminated by the specimen during inoculation, and the tubes should be decontaminated with sodium hypochlorite prior to being placed into the incubator. In a multi-tiered laboratory system, it is common to send samples from one laboratory to another for additional testing. If the sample is packaged properly and then repacked correctly, there is little risk. However, if packaged or repackaged improperly, diagnostic specimens can put package handlers, the public, and the receiving laboratory at significant risk. A set of international rules has been instituted to deal with the transportation of infectious materials (26). Transportation of diagnostic specimens (including any human or animal material which includes excreta, secreta, or blood and its components), tissues, or tissue fluids that are not reasonably believed to contain an etiologic agent does not require special packaging but must comply with requirements under OSHA’s blood-borne pathogen standard. Specimens reasonably expected to contain an etiologic agent need to be treated like an etiologic agent preparation. Leakproof packaging meeting minimum dimension standards and material standards consistent with Department of Transport and International Air Transport Association requirements needs to be used. It is the responsibility of the shipper to ensure that the package is safe and will not pose a risk to the receiving laboratory. Furthermore, laboratory directors should have ready access to the latest regulations, since they are subject to change from time to time. Risk Categorization of Microorganisms and Laboratory Design Classification systems have been developed as a method of categorizing the hazards associated with microbial agents and for clarifying appropriate procedures and facilities for working with them. All clinical laboratories should have a copy of the risk categorization of microorganisms (24) and Health and Human Services publications (13) as well as protective equipment and instructions on its use available for ready reference. Risk group 1 organisms are of low individual or community risk and are unlikely to cause disease in healthy workers or are not known to cause disease in healthy adults. Risk group 2 organisms have
4
JAMISON ET AL.
moderateor limited risk but are unlikely to be a serioushazard. Effective preventative and therapeutic measuresare readily available.Risk group 3 organismsare of high risk to workers and are capableof causingseriousinfection but tend not to be spread by casual contact. Risk group 4 organismsare of high risk, cause serious(often untreatable) infection, and also are readily transmitted by direct, indirect, or casualcontact. Clinical microbiology laboratories in most, but certainly not all, circumstancescan predict the microbial load being processedwith a reasonable degreeof reliability. Most sampleseither will be sterile or will contain only normal flora. Those that contain pathogensusually contain organisms that are characterized asrisk group 2. Occasionally, but not at an insignificant rate, sampleswill contain organismsclassifiedabove risk group 2, such as Mycobacterium tuberculosis or Brucella species.With that in mind, all clinical microbiology laboratories shouldbe capableof processing specimensat a minimumof containmentlevel 2 or biosafety level (BSL) 2. Clinical laboratoriesneed to be located a reasonabledistancefrom public areas,should allow limited public accessand have self-closingdoors, and should be outwardly designatedby a biohazard signwith appropriate information. All laboratory work surfacesshould be readily decontaminated. In addition to having hand-washing facilities and an accessiblebiosafety manual, the laboratories should be equipped with biological safety cabinetsand an autoclave or other acceptable method for decontaminationof waste. Personalprotective equipmentmustbe availableand easilyaccessible.
CUMITECH
29
associatedwith the various potential pathogens. Organic material such as serum,blood, or feces will interfere with the microbicidal activity of many agentsby acting asa physicalbarrier. In addition, organic complexing of chlorine- and iodine-containing compoundswill further reduce disinfectant activity. Other physical and chemical factors that will affect disinfection include pH, dilution, and water hardness.Alkalinity improves the activity of some-disinfectants,suchas gluteraldehydesand quaternary ammoniumproducts, while impedingothers,suchasphenols,hypochlorites, and iodine (44). For routine cleaning of benchtops,phenoliccompounds(0.2 to 3%), quaternary ammoniumcompounds(0.1 to 2%), and isopropyl or ethyl alcohol (70 to 85%) are the most effective and least noxious agentsavailable. Alcohols should not be usedin biological safety cabinets or where the potential for explosion exists. Sodium hypochlorite (1:lO dilution of household bleach) is used by many laboratories for routine disinfectionand is alsothe agentof choice for cleanupof spilled,potentially infectiousmaterials. If the spill is not too large, it can be covered with paper towelssoakedin bleach and then left for a minimumof 20 min to allow for inactivation, following which the spill materialscan be gathered for disposal.Larger spillsmay require preliminary wiping in order to reducethe organicload prior to disinfection.Protective outerwear (overalls,boots, etc.) shouldbe worn to precludecontaminationof clothing or shoes,aswell as a HEPA respirator. Microorganismsare inactivated by disinfectants at various rates, thus affecting the utility of a given combination. M. tuberculosis can be killed by iodophor (450 ppm) in 120to 180min, by substiAdministrative Responsibilities tuted phenol (3%) in 45 to 60 min, and by isoSafety practicesare a necessityfor all laborato- propanol (70%) in 5 min, thus making isoproparies. Since 1991, OSHA has inspectedphysician no1clearly the most effective surfacedisinfectant office laboratoriesto ensurecompliancewith the (50). blood-borne pathogen standard. The most comEquipment mon errors identified through the processinclude lack of (i) written hazard communicationpolicy or Aerosols are a potential risk associatedwith exposure control plan, (ii) material safety data openingof samplecontainers,centrifugation, vorsheets(MSDS) or chemical or biohazard labels, tex agitation, sonication,and openingof ampoules (iii) appropriate personal protective equipment of lyophilized bacteria. While droplets greater and adequateeye washstations,(iv) documented than 5 pm may not reach the lung, they are still paperwork for accidents, (v) fire and electrical hazardousbecausethey can contaminate the ensafety procedures,and (vi) an OSHA poster dis- vironment and laboratory workers’ hands. Laboratories shouldbe equippedwith biological safety Play (58). cabinetsand safety capped centrifugesto reduce Disinfection the risk of aerosolformation. Mechanical mixing Contaminationof the laboratory environment is of open tubes should be avoided. Small clinical a potential sourceof employeeinfection through laboratoriesmay not be equippedwith biological safety cabinets.As an alternative, technologists contaminationof skinor through other routes.All splashesand spillsneed to be cleaned up imme- working in thesefacilities shouldopen all sample diately. In cleaning up spills, one needs to take containersbehind plexiglasssplashguards. into consideration the volume of the spill, the The use of masksas an additional method of compositionand pathogenspresent,and the risks respiratory protection has been evaluated. Chan
CUMITECH
29
LABORATORY
and colleagues (16) observed that surgical masks were approximately 97% efficient, as opposed to the 99.9% efficiency of HEPA respirators. On the other hand, HEPA respirators are expensive (34) and require a snug and complete fitting to work properly. The use of HEPA respirators continues to be controversial in the management of aerosol containment. Furthermore, employees with chronic respiratory disease should be assessed medically to ensure that they are able to use a respirator. Hand Care Contamination of hands can be reduced by the combination of alertness and training, avoiding handling of contaminated materials, wearing gloves when handling patient specimens, and, in all circumstances, hand washing. Wearing gloves can prevent much hand exposure, but it is not infallible and, for some, is not in itself risk free. Gloves of vinyl composition are inexpensive but are prone to have holes or tear easily. Studies of gloves have demonstrated that a single pair of latex gloves is less likely to tear than either a single or a double pair of vinyl gloves; however, with latex gloves there is a significant potential for latex allergy, which can be significantly debilitating (17, 29). Reactions to gloves can be reduced by the use of powderless gloves or the use of liner gloves, although not without inconveniences or loss of sensitivity or dexterity. While laboratory coats, gowns, or uniforms are appropriate for BSL 2 work, for BSL 3 activity, it is recommended that a solidfront or wraparound gown be used. The material for the gowns must be impervious to accidental splashes or spills. These gowns should be of nonflammable material and should have arms with snug-fitting wrist cuffs. Frequent hand washing can lead to irritated cracked skin, which may provide an opportunity for infection to develop. Skin irritation can be reduced by the use of nondisinfectant soaps and nonionic skin lubricants. Contact Lenses and Bacteriology Laboratories While there is no epidemiological evidence to suggest that laboratorians are prone to develop Pseudomonas aeruginosa keratitis associatedwith contact lenses,Seal and Hay (46) point out that the microbiology laboratory is an environment with a potentially increasedrisk to the worker through contaminatedhands,splashing,or aerosolization. Microbiologists who wear contact lensesmust take additional care through routine useof goggles,daily washingof lensstoragecases in boiling water, and disinfection of lensesthemselves by heat or 2-h immersion in hydrogen peroxide. Microbiology laboratory workers should avoid 14-day-weardisposablecontact lenses,since retention of such foreign bodies coupled with
SAFETY
IN CLINICAL
MICROBIOLOGY
5
possiblecontamination may increasethe risk of keratitis. Sharps-RelatedInjuries Perhapsthe mostdangerousobjectsfor all health care workers, including laboratorians,are needles and other sharps.In 1951, Sulkin and Pike (53) reported more laboratory-acquired infections resulting from accidentswith needles and sharps than from any other factor. Through the decades, this relationship between accidents,sharpsexposures,and laboratory-acquired infections has remainedconsistent.Despite awarenessof the risks and hazardsassociatedwith sharpobjects,sharpsrelated injuriescontinueto occur frequently among phlebotomistsand bench technologists. Sharps-relatedinjuries continue to be a significant occupationalinjury in the health care setting. In clinical laboratories, the most frequent exposureto sharpsis amongphlebotomistswho handle sharpobjectssuchasneedlesattachedto syringes. Another sourceof exposureto sharpsis in needle aspirationfrom blood culture bottles.Broken glassware contaminated with live cultures can pose considerablerisk of infection or injury. Clinical laboratory injuries related to sharpscan be prevented through cautious phlebotomy technique and by ensuringthat needlesare safely removed from specimensbeing transported by syringe or using a one-handedrecapping technique. Avoidance of recappingof needles,proper handling of glassware,and careful disposalof broken glasswill all reduce the risk of laboratory-acquired infections. Laboratory personnelperforming animal studies with microbial agents are at special risk of accidentalinfection while trying to inject animals with M. tuberculosis (47), trypanosomes(41), plasmodia,Leishmania spp.,and Toxoplasma spp.(25). PCR At present,the AMPLICOR Chlamydia trachomatis test (42) is commerciallyavailable in many countries. The kit is based on three separate reactions: target DNA amplification, hybridization of the amplifiedproduct to a specificnucleic acidprobe, and detection of the amplifiedproduct by a calorimetric reaction. The test system is recommendedfor useonly with endocervicaland urethral swabsand urine from male patients. As receivedin the laboratory, the swabspecimens will contain only transport medium; the urine will be received in a native state. Urine is centrifuged to recover a cell pellet, which is then resuspendedin a “urine resuspensionbuffer,” after which the specimenis vortexed and allowed to incubate at room temperature. The specimenis then diluted and resuspendedby vortexing prior to amplification. Material originally obtained on swabs is simply diluted in “specimen diluent,” vortexed,
6
JAMISON
ET AL.
and incubated prior to amplification (42). The transport medium, in which the swabsare expressedafter collection, contains material which inactivates C. trachomatisand a wide variety of other microorganismscommonly found in the genital tract. The urine resuspensionbuffer also inactivates these microorganisms.Free nucleic acidsor ampliconsare regardedasnot biologically active and consequentlypose no biohazard (59). Therefore, the hazards associatedwith this and similarkits are thosewhich relate to causticand/or chaotropicagents.The packageinsert listssodium azide, magnesiumchloride, trishydrochloride,noninfectiousC. trachomatisDNA, EDTA, sodiumhydroxide, tetramethylbenzidine,and dimethylformamide, among other proprietary solutions (e.g., “detergent,” “ solubilizer”). Although a complete list of potentially hazardous agents cannot be compiled, those chemicals not considered innocuousare present in low concentrations, and Roche Diagnosticsconsidersthe kit nonhazardous in ordinary laboratory usage. Certainly the laboratory technologistwill need to wear a properly closed laboratory coat and gloves, but no specialprecautionsother than appropriate laboratory technique are required for safeuse of the kit. It is expectedthat nucleic acid diagnostickits, releasedin the future for general laboratory use by this or other commercial companies,will be configuredin a mannerconsistentwith safeusage in the ordinary clinical laboratory without use of biological safety cabinets. The development of “home brew” PCR technology in the clinical laboratory hasbecomewidespread.ThesePCR systemsposehazardsdifferent from those associatedwith the commercial kit currently available.The main differencelies in the processof detectionof the specificDNA amplicon which relatesto a positive test. In most cases,this is done through the use of agarosegel electrophoresisfollowed by stainingwith ethidium bromide or the use of a radioactive probe which specifically hybridizes with the target amplicon. Ethidium bromide is a highly toxic material, inducing acute eye and skin irritation as well as being a potent mutagen(31). The useof personal protective equipmentto protect eyesand mucous membranesis required when ethidium bromide is in use. Spills should be absorbedwith sand or vermiculite. Ethidium bromide-containingwaste shouldbe mixed with a combustiblesolvent and incinerated in an incinerator approved for use with toxic chemicals.Gloves should be changed after ethidium bromideuse.The biohazardsassociated with the use of radiochemicalsare manifold, and precautionsto avoid exposureof laboratory personnelmust be taken. A discussionof the precautionsto be observed, the appropriate techniques for use of these materials, and the subsequentdisposalof the radioactive waste is
CUMITECH
29
beyond the purview of this review. The reader is referred to the safety manualof his/herinstitution for local regulations concerning appropriate use and disposalof radiochemicals. Immunizations
HBV and M. tuberculosisare two agentsfrequently associatedwith laboratory-acquiredinfections. Immunization againstHBV is readily available and should be strongly recommendedfor individuals working with human blood or body fluids that can be potentially infectious. Efficacy studiesindicate that a regimenof three intramuscular dosesat the recommendedstrength of recombinant HBV DNA vaccine will produce an adequateantibody responsein greater than 90% of immunocompetentadult recipients (5). Postvaccination antibody testing is not routinely recommended,but it shouldbe consideredfor those at risk of needlestick accidentsthat might call for postexposureprophylaxis (19). Although OSHA hasdevelopedrulesand regulationsrequiring that vaccination be made available within 10 days of employment, experience showsthat across the health professionsvaccine acceptanceis only 40 to 80% (43).
BCG vaccine is derived from a strain of Mycobacteriumbovisoriginally attenuated by Calmette and Guerin. Although the vaccinehasbeenshown to have an efficacy of 80% in preventing pulmonary tuberculosisin children, there is continued uncertainty about the useof BCG. In 1988,its use in hospital workers was no longer routinely recommendedin the United Statesor Canada. Recent concern about the spreadof multidrug-resistant M. tuberculosisis cause to reexamine this policy. Detection strategies for Al. tuberculosis, including skin testing at regular intervals (6 to 12 months) followed by chest X-ray and giving prophylactic antibiotics to recent converters, may representa reasonablealternative for workers in mycobacteriallaboratories. Although typhoid fever isan uncommondisease in North America, on average400to 500casesare reported annually. Salmonellatyphi hasbeen recognized as a causeof laboratory-acquired infections, both from clinical specimensand from laboratory-derived materials. While typhoid vaccine is not routinely recommended,it should be consideredfor laboratory workers who routinely handle S. typhi or those who routinely work in enteric laboratoriesprocessingvery large numbers of specimensfrom travellers to countries with a high prevalence of S. typhi. Typhoid vaccine is availableboth in a parenteral heat-phenol-inactivated form (boosters are required every three years) and as an oral live-attenuated form. Although the oral vaccine can be usedas a booster alternative for the parenteral vaccine. there is no
CUMITECH
29
LABORATORY
information on booster requirements following oral immunization. Despite the frequent potential exposure to Streptococcus pneumoniae in specimensand cultures, laboratory workers are generally not at increasedrisk of pneumococcalinfections unless they are in a recognizedhigh-riskgroup. Pneumococcal vaccines are recommendedfor immunocompetent individuals who are at increasedrisk becauseof chronic illness,includingcardiopulmonary disease,diabetes mellitus, alcoholism, and cirrhosis.Additionally, it is recommendedfor immunocompromisedindividuals with splenic dysfunction or anatomicasplenia,Hodgkin’s disease, multiple myeloma,chronic renal failure, and nephrotic syndrome.Personswith symptomaticand asymptomatichuman immunodeficiencyvirus infection shouldalsobe vaccinated. Specialimmunizationsmay be required if handling of certain pathogenssuchasS. typhi, Yersinia pestis, Vibrio cholerae, and/or arbovirusesoccurs regularly. The advice of a specialistshould be sought,and a site-specificpolicy shouldbe devised on the basisof the nature of the work and an assessment of the risk. Underlying
Illness and Other Factors
Certain individuals may be at increasedrisk of infection becauseof significantunderlying conditions, suchaspsoriasis,open skin lesions,chronic respiratorydisease(suchascystic fibrosis),steroid therapy, splenectomy,immunosuppressivedisorder, or human immunodeficiencyvirus infection. Suchindividuals may need to consideradditional measures to precludelaboratoryinfections.In some instances,a vocational changemay be a serious option. All healthy adultsworking in microbiology laboratories should maintain adult immunization schedulesagainst tetanus and diphtheria. Although there is no indication for the regular usage of other vaccines,workersin specializedlaboratories may consider the use of vaccines against cholera and meningococciin specificsituations. Personal
Work Habits
Well-documented laboratory-acquired infections can often occur with no obvious break in technique,especiallywith microorganismsof high virulence and with low inoculum demands(5, 8). Often infectionsoccur when the degreeof danger is not appreciated.A caseof laboratory-acquired melioidosisin which the organismwas misidentified as Pseudomonas cepacia was documented (45). Infection with Brucella melitensis, misidentified as Moraxella phenylpyruvica, has also been documented(7). All specimensmust be handled as if they contain pathogensthat are potentially dangerousto personnel.This includes not only clinical samplesbut materialsfor quality control
SAFETY
IN CLINICAL
MICROBIOLOGY
7
as well (8). There are specific technical caveats, suchas avoidanceof mouth pipetting, safeuse of centrifuges, use of aerosol containment, and avoidanceof splashing,which bear emphasisand which must be enforced. The potential existsfor laboratory reagents to cause chemical injury to skin or even to contain unknown pathogensin the caseof human or animalbiological materials. Thus, direct contact between these reagentsand the skin or mucosashould be avoided by using careful technique; the use of glovesis necessary. BACTERIA Disposal
of Biological
Waste
All wastematerial leaving microbiology laboratories should be considered infectious and, as such,shouldbe decontaminatedor sterilized prior to leaving the laboratory. For cultures and specimens,this is usually done by autoclaving. Large volumes of serum and urine may be disposedof directly through the sanitary sewer system,provided that they are flushed through with a sufficient volume of water. In the event that a proper disposalsystemis not available, these materials shouldalsobe autoclaved. Specimencontainers,secondarytransport bags, swabs,wastewashsolutionsfor assays,etc., are all to be consideredcontaminatedwaste. Suchwaste must be disposedof in a manner appropriate for each individual category (27, 33). Swabs,applicator sticks, etc., removed from clinical specimens during processingshould be placed immediately into covered metal panslocated insidea biological safety cabinet or into vesselscontaining a suitable disinfectant. At suitableintervals, thesematerials should be collected, carried directly to an autoclave, and sterilized before being placed into plastic bags or unmarked containers for later disposalasnoninfectioustrash. Reusablepipettes should be stored in pipette jars containing disinfectant after use.At suitableintervals,the pipettes shouldbe collected in metal containersand autoclaved. Disinfectant solutionsshouldbe changed daily. Spent disinfectantsolutionsshouldbe autoclaved prior to being discardedinto the sewer.If the disinfectant used is a sodium hypochlorite solution,it shouldnot be autoclavedbut shouldbe changed frequently. Waste disinfectant can be washedinto the sewersystem.Animal carcasses, body fluids, and bedding that have been exposed to infectious agentscan be disposedof by incineration. (Usually, the laboratory is not involved in disposalof the bedding,food, and/or cageusedto contain infected animals,asit is the responsibility of the animal caretakers.) Contaminatedlaboratory trash containing largevolumesof liquid (e.g., wash solutions,used buffer, tissueculture fluids, etc.) is best decontaminatedby autoclaving prior to disposal.Large volumesof contaminatedplas-
8
JAMISON
ET AL.
ticware or wastepaper products should be autoclaved (best) or collected for incineration. Such trash shouldbe placed in a rigid plastic container or in a rigid cardboardcontainer lined with plastic. The containersshouldbe labelledto indicate that they contain contaminated and infectious waste and that they are to be incinerated without opening. The laboratory should cooperatein developing proceduresto be followed in the event of an accident involving potentially infectious waste prior to incineration. All specimensfor stainingand culture received in the mycobacteriologylaboratory shouldbe documented,handled, and cultured asif they contain A4 tuberculosis (54). The outside of transport containersmay be contaminated, thus posing as much of a health risk as the sampleinside (2). Sputum samplesshouldbe transported in a manner that is safe for the courier and receiving laboratory. A sealedplasticbag with a sidepocket that allows the requisition to be attached but separatedprovides safety. Extrapulmonary samplesmay alsobe positive and therefore shouldbe viewed asa hazard to laboratory personnel(1). All personnelreceiving samplesshouldwear gloves, gowns(lab coat), and masks.All containerswith visible soilingon the outsideshouldbe decontaminated with a phenolic wipe. All sputumcontainersmustbe openedunder a properly working class II biological safety cabinet. Laboratories should monitor the numberand originsof leakingsputum containersso that the submitterscan be notified. Receipt of leaking containers from multiple clients may indicate a fault in the container design and would necessitatea change to ensurestable container walls and a securescrew-oncap. While specimen preparation can be done in BSL 2 facilities, propagation and manipulation of cultures of M tuberculosis shouldbe performed only in facilities capableof supportingBSL 3 practices. Previously skin test-negativeemployeesin mycobacteriologylaboratoriesshouldbe given a tu-’ berculin skintest semiannually.This schedulemay increaseif a skin test conversion occurs in any employeewithin the unit. All employeesshowing conversionsmust be reported to public health authorities and directed to appropriate medical care. There is no needfor BCG immunization for laboratoriansworking within efficiently operating units. Of the bacteria that are isolatedin mostclinical laboratories,M tuberculosis is the predominant speciesrequiring BSL 3 facilities. Other risk group 3 bacteria, suchasFrancisella spp., Y pestis, and Bacillus anthracis, are more rarely encountered. Specimenssuspectedof containing such bacteria shouldbe referred to an appropriately equipped referencelaboratory or processedin BSL 3 facilities in-house.
CUMITECH
29
FUNGI The dimorphic fungi, particularly Coccidioides immitis, presenta potential safety hazard. The risk of infection through inhalationof aerosolizedconidia or through skin puncture by contaminated sharpsdemandsstrict observanceof safety precautions.Avoidance of infection requirescareful microbiologicaltechniqueaswell asawarenesson the part of employeesof the nature of the risk not only to themselvesbut also to colleaguesin the laboratory and even to personneloutside the laboratory. BSL 2 facilities are recommendedfor most mycologicalpractice. However, in view of the high infectivity of C. immitis and the fact that asmany as 90% of infected laboratory workers exhibit clinical disease(61) it is recommendedthat BSL 3 facilities be usedfor work with this organism.A similar recommendation applies for processing culturesof Histoplasma capsulatum and Blastomyces derma titidis.
In the event that a laboratory employee is immunosuppressed, it is recommendedthat work with organismsof low infectivity, suchas Cryptococcus neoformans andAspergillus spp.,be carried out in BSL 2 facilities. Employeesworking with thesefungi shoulduse personal protective equipment, including gowns, masks,safety glasses,footcovers, and respirators. VIRUSES Cell Culture Primary tissuecultures prepared from monkey kidneys are known to be infected by a large number of endogenoussimianviruses.Theseendogenous viruses, some of which are latent, include membersof the Papovaviridae, Adenoviridae, Poxviridae, Picomaviridae, Reoviridae, rotaviruses,Orthomyxoviridae, Pseudomyxoviridae, and Retroviridae. Although mostsimianvirusesare considered to be of no clinical importance to humans,the potential for laboratory-acquired infection with one or more of these agentsis real (4). The infection may be severe and/or life-threatening in humans.SimianherpesvirusB, a latent infector of monkeys,displaysrelatively low pathogenicity in the monkey. However, it is a highly dangerous pathogen to humansand may be fatal. Furthermore, animal products (cells, tissues,serum,and blood) from a variety of nonprimate species(e.g., rodents,birds, and cattle) have been shownto be contaminatedby a variety of latent viruses.Latent virus infections in primary cell cultures often are not recognized.Therefore, all primary tissuecultures, regardlessof their origin, shouldbe considered to be a potential source of laboratory-acquired infection and treated accordingly (59). The variety of biological materials used for either tissueculture growth and maintenanceor
CUMITECH
29
LABORATORY
virus isolation and identification renders a specific discussion of each of the potential hazards impossible. It will suffice to simply remind the reader of the potential for inapparent infection in seemingly healthy donor animals. Any material containing animal products (e.g., cell or organ cultures, erythrocytes suspended in buffer, serum as a supplement in tissue culture medium, and antibody prepared for specific virus identification) should be considered to harbor adventitious agents such as viruses, mycoplasmas, or other bacteria and should therefore be considered a source of infection for laboratory personnel. All materials should be handled in an appropriate biological safety cabinet whenever possible. Appropriate laboratory techniques and personal safety equipment combined with adequate facilities for preparation and utilization of the materials should obviate any real hazard. The importance of education of laboratory personnel to the potential of these products as a source for laboratory-acquired infection cannot be overstressed. Preparation of the Specimen for Inoculation It is assumed that all preparative techniques are performed in a biological safety cabinet (see below) to prevent contamination of the environment. The formation of aerosols from materials containing infectious agents has the potential to cause multiple infections. Even with the use of appropriate containment, the most common hazards are associated with aerosol formation during centrifugation (13, 33), grinding, vortexing, and pipetting. In a sense, it can be assumed that these procedures are at best “controlled spills” (4, 56), and measures must be available to contain the “spill” and to decontaminate the area when the procedure has been completed. The technologist should have available an effective disinfectant (e.g., sodium hypochlorite solution) which can be utilized immediately without breaching the containment barrier. If, for instance, centrifugation cannot be performed in a barrier-protected space, the manufacturer’s recommendations for safe use of the centrifuge should be followed without deviation. In most cases, this requires that the potentially infectious material be placed in a sealed tube and inserted into a centrifuge safety carrier prior to centrifugation. The centrifuged material must be opened only in a barrier-protected area once centrifugation has been accomplished. Vortexing of a liquid specimen (urine, respiratory secretions, lavage fluid, etc.) or a specimen (swab) to which liquid has been added should be done only in a tightly sealed tube. Prior to vortexing, any specimen received in the laboratory which was not contained in a screw-capped tube should be transferred into one by pipetting; decanting should be avoided to reduce the risk of spillage. Obviously, such a transfer should be made only in
SAFETY
IN CLINICAL
MICROBIOLOGY
9
a biological safety cabinet. The method of enclosing the specimen, still in its secondary container, inside another sealable transparent plastic bag is acceptable but cumbersome. In any case, after being vortexed, the specimen should be inverted slowly so that air mixes with fluid to resorb aerosolized particles. Whenever practical, the containers should be allowed to stand before being opened to let aerosols settle. Those specimens which are received on a solid support (swabs, stick, or disposable loop) must be soaked in liquid prior to vortexing. After vortexing, the aerosol must be allowed to settle and the support must be removed from the container with forceps and discarded into a vessel containing disinfectant. Grinding of tissues from autopsy or biopsy and mixing of stool suspensions also potentially create aerosols. Dangers can be minimized by enclosing the mortar and pestle, grinding tube, stool cup, etc., in a transparent plastic bag to minimize potential contamination of the barrier space. The use of disposable tissue grinders, which are fashioned with a transparent safety shield (bag), is helpful. If electric grinders are utilized, the use of slow speeds will minimize aerosol formation and splashing. The most common hazard associated with pipetting procedures involves the use of mouth suction, a technique that must be strictly forbidden. The aerosol hazard associated with pipetting can be reduced by the use of proper technique, biological safety cabinets, and protective clothing. However, the potential hazards associated with oral ingestion can be eliminated completely by the use of mechanical pipetting aids; therefore, mechanical pipetting aids must be mandatory for all pipetting procedures. Additional hazards are created by liquid dropping from a pipette to a work surface, by mixing cultures by alternate suction and blowing, or by forceful ejection of an inoculum by blowing out the last drop. Proper pipetting technique, then, will avoid these hazards. The use of pipettes calibrated to deliver to the tip avoids the problem of forcibly expelling the last drop of a liquid from a pipette tip. The use of gravity flow to mix liquids should be a standard procedure, minimizing hazards (bubbles and splashes) created by mixing of liquids by alternate suction and blowing with the pipette. All pipetting should be performed over a disinfectant-soaked towel to catch any fallen drops. Pipettes should be disposed of by either immersing them in disinfectant or placing them in pans suitable for autoclaving. The processing of blood or blood products as specimens from which infectious agents may be isolated should not pose problems when reasonable laboratory precautions are followed. As always, “universal precautions” apply (11). The potential for laboratory acquisition of human immunodeficiency virus, HBV, or hepatitis C virus is
10
JAMISON
ET AL.
real. Some infectious virus should be presumed to be present in all specimens which contain blood or which are contaminated with blood. The use of personal protective equipment at all times when working with specimens received in the virus laboratory, the strict observance of rules which pertain to opening of potentially infectious specimens in biological safety cabinets or with other barriers, and the processing of such specimens in an appropriate manner should preclude infection of laboratory personnel. Inoculation of the Specimen into Cell Tissue Culture The inoculation of cell and/or tissue cultures with potentially infectious agents poses no hazard not mentioned above. Specifically, almost all clinical laboratories will perform this operation by pipetting. No specific hazards are attendant; pipetting is to be done by use of a mechanical pipetting aid, paying reasonable attention to the precautions previously mentioned. However, excessively mucoid specimens may pose additional difficulties. Splashes, mucous drips, etc., may accidently contaminate the outside of tissue culture tubes. Disinfectant for decontamination of the floor of the biological safety cabinet and the outside of the tubes if necessary should always be available to the laboratory technologist. Inoculation of Embryonated Eggs or Laboratory Rodents Animal inoculation is not routine in most of the clinical laboratories in North America. However, state or regional public health laboratories may, on occasion, utilize rodents for isolation of potentially rare, fastidious, or exotic viruses. Egg inoculation for isolation of influenza virus from respiratory secretions is more common, although not routine in most clinical laboratories. The major danger inherent in both procedures involves the potential for self-inoculation by means of needle sticks or accidents with other sharp objects, such as scalpel blades, capillary pipettes, etc. Laboratory workers should use extraordinary care to prevent puncture injuries to hands and fingers from sharp instruments or devices during inoculation of animals or at subsequent autopsy and biopsy. After use, disposable syringes and needles, scalpel blades, capillary pipettes, or other sharp items should be placed in puncture-resistant containers for subsequent autoclaving and disposal. Needles should not be recapped, nor should they be purposefully bent, broken, or removed from disposable syringes (35). Inoculation of the animals or eggs should occur in a biological safety cabinet. Inoculated eggs should be sealed with tape or glue prior to incubation. Inoculated animals should be returned to the animal quarters and housed in animal BSL 2. Tissues from coma-
CUMITECH
29
tose humans must be considered to be infected with rabies, even in the absence of known exposure. The animals and their excretions and tissues are potentially infectious. Practices and facilities compatible with BSL 2 are considered to be adequate to protect personnel in the laboratory and animal quarters (13). Reaction with Antibody of Known Specificity The identification -of tissue culture isolates by neutralization with known antibody has been the mainstay of the virus laboratory (4). The test consists of mixing the unknown clinical isolate with a dilution of known antiserum, allowing time for the antigen-antibody reaction to occur (thereby forming the neutral mixture, if it is to be formed), and then inoculating sentinel tissue cultures with the postulated neutral mixture. The lack of development of cytopathic changes in the inoculated cultures is indicative of neutralization and therefore identification of the isolate. The laboratory safety issues inherent in this procedure are the same as those involved in inoculation of tissue culture with the clinical specimen from which the isolate was derived. The isolate is potentially infectious; it should be handled in a biological safety cabinet. Precautions taken to minimize aerosol formation during centrifugation, pipetting, etc., must be observed. As all serum samples utilized in these tests by routine clinical laboratories are standardized and obtained from either reputable commercial sources or governmental agencies (CDC, the National Institutes of Health, etc.), the probability that they contain adventitious viral agents is minimal. Nonetheless, they should be handled with due precautions. Care must be taken to avoid needle sticks when rehydrating ampoules, and spills should be wiped up with suitable disinfectants. It is important to emphasize that a putative neutral mixture may contain nonneutralized virus and should always be considered infectious. In the relatively recent past, methods developed for the rapid and direct detection of viruses in clinical specimens have been adapted for use in the identification of viruses isolated by inoculation of tissue cultures with clinical specimens. In these systems, the tissue cultures essentially function as “amplification systems” and are used to increase the amount of viral antigen to a level consistent with the sensitivity of the detection method used. The enzyme-linked immunosorbent assay (ELISA) and latex agglutination test are most often encountered in the routine clinical laboratory. All of the procedures used in these assays are standardized, and each of the commercially available assays has been approved for use by the U.S. Food and Drug Administration or its equivalent. Therefore, each carries a package insert giving precautions and indicating the hazards associated
CUMITECH
29
LABORATORY
with the specific test system. Such issues as the potential for chemical burns by acids or alkali and the potential for induction of hypersensitivity by reagents used in the test, the biohazard associated with collection, and the processing of patient specimens are addressed. Laboratory personnel must be made aware of these issues and must follow the recommendations when testing both clinical specimens and tissue culture isolates. In each assay, the viral isolate (or virus contained in the clinical specimen) to be tested is “native,” that is, it is not treated with any physical or chemical agent that might inactivate it. It is therefore fully infectious, and care must be taken to handle it in an appropriate manner. Personnel should always wear personal protective equipment, and all procedures involving the native isolate should be performed in the biological safety cabinet. Once the infectious material has been added to the assay system (tube, card, etc.), subsequent steps in these assays can be performed outside of the biological safety cabinet, provided that they are performed on a disinfectant-soaked towel and that more disinfectant is readily available. The use of disposable plastic transfer pipettes should be encouraged. Care should be taken to preclude aerosol formation, and spills should be disinfected and wiped up immediately. The identity of many viral isolates may be uncertain, even given details such as source and type of specimen, clinical history, type of cell culture in which it was isolated, cytopathology, etc. Viral isolates are frequently identified by specialized procedures which may use only one or at most two antigen-antibody systems: hemagglutination inhibition, complement fixation, and direct immunofluorescence. Each of these procedures carries with it an intrinsic minimal risk, such as the use of animal products and volatile fixatives, and the dangers associated with it should be made known to the staff. In each procedure, the major biohazard is the viral isolate itself. Secondary passages which are necessary either to obtain enough of the virus for testing or to obtain infected cells must be performed in a manner analogous to the processing of the original clinical specimen, with due attention to centrifugation, pipetting, and use of personal protective equipment. In each procedure, the initial specimen is infectious until possibly fixed during the procedure, such as in immunofluorescence. Electron Microscopy Direct examination of clinical specimens by electron microscopy for morphologically identifiable virus particles is a rapid and useful diagnostic technique, particularly in the diagnosis of viral enteritis (3, 48). As ordinarily practiced, a stool suspension (5 to 10%) in buffer is prepared, mixed with 1.5 to 3% phosphotungstic acid or any other electron-dense stain, and applied to a plastic-
SAFETY IN CLINICAL
MICROBIOLOGY
11
coated copper grid used for electron microscopy. The mixture is allowed to adhere for 1 to 2 min, after which the grid is blotted dry. If the stool suspension is particularly turbid, it may be clarified by low-speed centrifugation prior to preparation for the electron microscope or the stained grid may be rinsed briefly after preparation. The stained specimen is subsequently examined in the transmission electron microscope. Opportunities for infection of laboratory personnel primarily relate to preparation, use, and disposal of the clinical specimen, so appropriate precautions, including the use of personal protective equipment, must be observed. The possibility of aerosol formation during centrifugation must be kept in mind. Further, if the grid has been rinsed during preparation, the rinse fluid and blotting paper must be considered contaminated and disposed of properly. Although the potential for laboratory infection is remote, it must be appreciated that stained virus on the grid is still biologically active (3) unless or until inactivated by osmium vapors, formaldehyde, glutaraldehyde, or some other fixative. UV radiation (900 pW/cm2) at 15 to 20 cm for 10 min has been used to inactivate virus on stained grids (18). Other suitable disinfectants (e.g., dilute sodium hypochlorite) can be used to wipe up spills, sterilize forceps, etc. Electron microscopic examination of sections of tissues obtained at biopsy or autopsy is performed only infrequently as a service of the diagnostic laboratory per se. Because of the specialized nature of the preparation required, it is usual that this service is considered to be a separate and distinct function of other sections of the hospital laboratory or pathology service. The volatile fixatives, polar reagents, and resins used in preparation of specimens for transmission electron microscopy are toxic and may be environmentally dangerous. A discussion of these hazards is beyond the scope of this document, and the reader is referred to standard textbooks relating to the technique of electron microscopy (28). Immunofluorescence Rapid identification of viruses in clinical specimens can be accomplished by direct staining with antiserum conjugated with fluorescein isothiocyanate or a variety of other labels detectable by histochemical staining. Specimens may be received in the laboratory smeared on glass slides and may be either fixed or unfixed. Alternatively, they may be secretions, scrapings, or tissues contained in tubes in a native state. It is important to recognize that all unfixed materials are potentially infectious. Laboratory personnel involved in the processing of these specimens should wear personal protective equipment. If specimens are prepared in the laboratory, they must be air dried in a biological
12
SAMISON
ET AL.
safety cabinet prior to fixation with acetone, methanol, or other polar solvent that renders them noninfectious. However, such fixation should be done in a biological safety cabinet over a towel soaked in disinfectant. Forceps, slides, transfer pipette, swabs, etc., are all considered contaminated and should be disinfected immediately after use. Overtly contaminated materials should be autoclaved prior to being discarded. Examination of the stained specimens in a fluorescence microscope potentially exposes the technician to a source of UV light. It is important that the microscope be in proper working condition and that the UV light source be shielded appropriately in order to avoid cornea1 and skin damage.
CUMITECH
29
and responsiblemanner. A properly closedlaboratory coat and glovesshouldbe worn at all times while performing the ELISA, first, to protect the individual from inadvertent infection and second, to precludechemical-inducedburns or skin irritation. If used as suggestedin the package insert, theseassaysare consideredto be low risk. Containment
in the Clinical Laboratory
Virology
The issueof appropriate containmentof infectious agentsin clinical specimensis critical in clinical virology, particularly consideringthe implications of laboratory infection with exotic, highly contagious,and potentially fatal viruses(15). The CDC publishedan extensive monographon bioELISA safety (13), which includesa discussionof laboraELISAs are generally specific“capture” assays, tory biosafety level criteria and biological safety wherein a specificantibody is immobilized onto a cabinets. For the convenience of the reader, a solid support system (tube, well, or polystyrene table from that documentwhich summarizesrecbead). The clinical specimenis brought into con- ommendedbiosafetylevelsfor infectious agentsis tact with the support, and any specific antigen reproducedhere (Table 1). capableof reacting with the antibody is immobiThe essentialappropriatenessof these guidelized on the support by the antigen-antibody re- lineshasbeenaffirmedrecently in the publications action. After nonreactivematerial iswashedaway, of two national review groups,one in France (57) a secondspecific antibody, labelled with an en- and the other in Japan (49). It is, however, a zyme capableof being detected in a calorimetric truism that none but the most elaborate clinical assay,is added to the test systemand allowed to laboratory will have containment facilities suffireact. After further washing,an assayfor the en- cient for work at BSL 3 and 4. However, almostall zyme is performed.A positive test for the enzyme clinical virology laboratories will have facilities (i.e., development of color) is indicative of the compatiblewith BSL 2. This level of containment presenceof the specific antigen in the original is suitable for work with agents of moderate clinical specimen.The primary hazardsassociated potential hazard to laboratory personneland the with this procedure relate to processingof the environment. Standard laboratory practice in the original clinical specimen,which is potentially clinical laboratory ensuresthat manyof the special infectious.Almost all of theseproceduresrequire practicesandlaboratory facilities dictated for BSL that the original specimenbe vortexed and then a 3 facilities will, in fact, be mandatory in BSL 2 samplebe addedto the solid support. Obviously, laboratories.Nonetheless,it is inappropriate and aerosolformation is of concern. The above-men- unsafeto producelargequantitiesof concentrated tioned proceduresfor minimizing hazardsassoci- infectious virus in the clinical laboratory (14). ated with aerosolsare pertinent here. Ideally, the Discussionof the physicaldesignof the laboratory mixed specimen should be transferred to the and regulationof laboratory personnelin research reaction vessel by a technician equipped with facilities which processlarge volumes of concenpersonalprotective equipmentworking in a bio- trated infectiousvirus in a mannerthat potentially logical safety cabinet. At a minimum, this can be may produce aerosols,spills,and other accidents accomplishedin a semienclosed space(hood) over is beyond the scopeof this paper. It is significant a towel soaked in disinfectant. Several of the that recent reports of laboratory-acquired virus commerciallyavailableELISA kits are configured infectionsin BSL 3 containmentresearchfacilities such that an inactivating agent is present in the stressthe responsibilityof individual personnelin collection kit and consequentlythe specimenis acquisition of the infection (needle stick, lack of inactivatedduring transport to the laboratory. The latex gloves) (12, 35). These reports emphasize biohazard risk of these specimensis minimal. that while physical facilities appropriate to the Reagentsusedin the assayfor the enzymemay be hazard level are a necessity, laboratory safety dangerous.Although the kits are deemedsafe in requiresthat personnelboth understandthe prinordinary usage,each containsmaterialsthat may ciples and practicesinherent in laboratory safety induce skin irritation and damage to mucous and avail themselvesof provided personalprotecmembranes.Several kits contain materials that tive equipment.Proper useof containment faciliare known to induce delayed hypersensitivity in ties implies education of laboratory personnel, lower animalsand that are mutagens.The labora- and such education should be of the highest tory technician must usethese reagentsin a safe prioritv.
CUMITECH
29
LABORATORY TABLE
BSL
Agents
1
Not known to cause disease in healthy adults Associated with human disease; hazard = autoinoculation, ingestion, mucous membrane exposure
2
3
1. Summary
SAFETY
of recommended
BSLs
Standard microbiological practices BSL- 1 practice plus: Limited access Biohazard warning signs “Sharps” precautions Biosafety manual defining any needed waste decontamination or medical surveillance policies l
l
l
Indigenous or exotic BSL-2 practice plus: agents with potential for Controlled access aerosol transmission; Decontamination of all disease may have seriwaste ous or lethal conseDecontamination of lab quences clothing before laundering Baseline serum l
l
l
l
for infectious
MICROBIOLOGY
None required
13
agentsU
Safety equipment (primary barriers)
Practices
l
IN CLINICAL
Facilities (secondary barriers) Open bench top sink required BSL-1 plus: autoclave available
Primary barriers = class I or II BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials; PPE: laboratory coats, gloves, face protection as needed Primary barriers = class I BSL-2 plus: or II BSCs or other physPhysical separation ical containment devices from access corridors used for all manipulations Self-closing, doubleof agents; PPE: protective door access lab clothing, gloves, respiExhausted air not ratory protection as recirculated needed Negative airflow into laboratory Primary barriers = all proBSL-3 plus: cedures conducted in Separate building or class III BSCs in combiisolated zone nation with full-body, air= Dedicated supply/ supplied, positive presexhaust, vacuum, and sure personnel suit decontamination systems Other requirements outlined in the text l
l
l
l
4
Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission
BSL-3 practice plus: Clothing change before entering Shower on exit All material decontaminated on exit from facility l
l
l
l
l
a BSC, biological
safety cabinet;
PPE, personal
protective
Although there is no written policy of the CDC which specifically delineates those - situations in which involvement of the local clinical laboratory in specific viral diagnosis is less appropriate, certain clinical situations may require consultation and collaboration with the CDC. The Special Pathogens Laboratory has indicated that the CDC will respond to requests for procedural assistance in specific cases where potentially highly communicable exotic viruses are suspect. It is suggested that the CDC be consulted and that clinical specimens be collected by CDC personnel and transmitted by them to the CDC for processing. However, potentially infected tissue samples may be handled in BSL 2 facilities in accordance with BSL 3 practices. Universal precautions should be followed whenever human blood is handled (11). PARASITES General Safety Precautions All samples and specimens must be regarded as potentially hazardous. Blood, feces, other body fluids, and certain parasite cultures (e.g., those obtained by the Baermann and Harada Mori techniques) may be infective to laboratory personnel. Since many parasitic infections are acquired
equipment.
via the oral route, universal precautions must be followed at all times. In many procedures a biological safety cabinet is not necessary; however, a cabinet should be used whenever there is a potential for aerosolization or droplets, such as in procedures involving blending, sonicating, and vigorous shaking or mixing. Eggs of Ascaris spp. will remain viable and embryonate, even in formalin or sodium acetate-acetic acid-formalin. When handling fresh fecal material, laboratory personnel should be aware that certain forms of many protozoan species, e.g., cysts of Entamoebahistolytica and Giardia spp., oocysts of Cryptosporidium spp., eggs of pinworms (Enterobiusvernzicularis), tapeworms (Taeniasoliumand Hymenolepis nana), and the larvae of Strongyloidesstercoralis,as well as other microorganisms, may be infective. Biological safety cabinets should be used when handling and processing blood and other unpreserved body fluids. Classification According to the Degree of Risk Classifications based on the pathogenicity of the parasite, the mode of transmission and host range, the availability of effective treatment, and preventive measures have been prepared and published
14
JAMISON ET AL.
CUMITECH
TABLE Organism Acanthamoeba spp. (especially A. culbertsoni) Angiostrongylus spp. Ascaris lumbricoides Babesia spp. Balantidium coli Brugia spp. Capillaria spp. Clonorchis sinensis Cryptosporidium spp. Dipetalonema spp. Diphyllobothrium spp. Dracunculus medinensis Echinococcus spp. Entamoeba histolytica Enterobius vermicularis Fasciola spp. Fasciolopsis buski Giardia lamblia
Hookworm species spp. spp. (mammalian)
Hymenolepis Leishmania
29
2. Parasites of risk group 2” Comment G,
A
Organism Loa loa Mansonella
Comment
ozzardi
Microsporidia Naegleria spp. (especially N. fowleri) G
G c G G
G G
Onchocerca volvulus Paragonimus spp. Plasmodium spp. (human and Pneumocystis carinii Schistosoma spp. Strongyloides spp. Taenia spp. Toxocara canis Toxoplasma gondii Trichinella spp. Trichomonas vaginalis Ttichostrongylus spp. Ttichuris trichiura Trypanosoma brucei subsp. TIypanosoma cruzi Wuchereria bancrofti
simian)
G, C
G* G C
G G C X
C, G
G
a C, cabinets required for aerosol containment; G, gloves required; X, may be teratogenic; G*, caution-thick blood films are unfixed and may contain highly infectious pathogens; A, avoid aerosolization, work in cabinet.
in the United Kingdom (55), United States(13), and Australia (51) and by the World Health Organization (62). On the basisof theseclassifications, all parasitesare consideredto belong to risk group 2 during their infective stages. Risk group 2 microorganisms includepathogens that can causehuman,plant, or animaldiseasebut are unlikely to be a serioushazard to laboratory personnel,the community, livestock, or the environment. Laboratory exposuresmay causeserious infection, but effective treatment and preventive measuresare available and the risk of spreadis limited. Table 2 liststhe parasitesincluded in this category. NONINFECTIOUS HAZARDS Fire Hospitalsand other major health care facilities usually have well-developedfire prevention policies aswell as proceduresto be followed in the event of fire. In addition to general awarenessof fire regulations,such as interpretation of alarms, knowledgeof escaperoutes, andprecautionsto be taken on leaving, the microbiologyworkers should alsobe familiar with the potential fire hazardsin their area. Microbiology laboratories may have nakedBunsenflames,and there is alwaysa risk to personalsafetywhen handlingflammables.Moreover, such flammablesshould be stored on site in minimal volumes and in fire-proof cabinets. Workers shouldbe aware of the location of fire alarms, switches, and extinguishers, and these items should be checked on a regular schedule.
Fire doors should not be wedged open. Large institutions often have fire wardensdesignatedfor each floor; these personstake control of occupants on their floor and ensuretheir safe egress. There shouldalsobe-personneltrained in the use of fire extinguishers.Fire drills at regular intervals shouldbe part of the safety program. Electrical Hazards Only approved electrical equipment should be used. Damaged electrical insulation should be replaced. Extension cords should not be used. Appliances should be grounded, annual grounding checksshould be performed, and noise- and heat-generatingequipmentshouldbe kept in separate rooms. Gas Cylinders Gas cylindersare generally safeaslong asthey are not damaged.If a cylinder of compressedgas falls, snapsoff a regulator, and losesgasrapidly, the cylinder becomesan airborne torpedo that can inflict seriousdamageto humansand to the laboratory. Thus, compressedgascylinders must alwaysbe chainedto a solidstructure, suchasa wall, or supportedby free-standingbraces.Safety caps must be in place when the cylinder is being transported or when it is not in use. Chemicals Many of the reagentsemployedin the microbiology laboratory are consideredto be hazardous and mav fall into one of the following categories:
CUMITECH
29
LABORATORY
corrosive, toxic, carcinogenic, flammable, and explosive. It is now a legal requirement that all purchased chemicals that may be hazardous be accompanied by MSDS and that the chemicals be labelled in a simple format that will warn persons of the potential hazards. MSDS must be maintained in the work area in which the materials are used. They must be kept up-to-date, and all laboratory workers must be familiar with their location and their use. Compounds must be correctly labelled, including the use of hazard codes and labels as appropriate. Chemicals should be stored and secured in a safe manner. Flammables, corrosives, and compressed gases should be stored separately, preferably at floor level, to avoid accidents resulting from falls. Equipment and instructions should be readily available and understood in order to clean up any spills. The instructions for each type of spill-acid, alkali, flammables, mercury, or other noxious material-should be documented in the safety manual. A copy of the relevant section should be available where the items are stored. Protective clothing, gloves, and goggles should be available. All laboratories should have emergency showers and clean eye wash stations. In the event of a spill, the nearest employee should call for assistance, and the laboratory safety officer or supervisor should assume responsibility for ensuring safe cleanup. Noninfectious Waste The disposal of chemical waste in most clinical laboratories associated with hospitals, public health establishments, medical schools, etc., occurs under the auspices of a branch of the administration specifically charged with this matter. The MSDS which accompanies each potentially hazardous chemical in North America suggests waste disposal methods for each specific chemical. These form the basis for general regulations concerning disposal of nonradioactive chemical waste. It is the responsibility of the laboratory to be aware of the general problem of chemical waste disposal, to recognize those chemicals which require specific methods of disposal, and to conform to those specific regulations developed by its parent organization for chemical waste disposal. A general review of these procedures is outside the scope of this Cumitech. Radioactive waste disposal is subject to various local and national legislated restrictions. Obviously these should be followed strictly. Preservatives Two of the most frequently used preservatives contain reagents which call for adequate waste disposal and safety precautions: mercuric chloride in polyvinyl alcohol (PVA) and formaldehyde in sodium acetate-acetic acid-formalin.
SAFETY
IN CLINICAL
MICROBIOLOGY
15
Special safety considerations Formaldehyde. Because of the potential carcinogenicity and toxicity of formaldehyde, regulatory boards in different countries have established requirements regarding the levels of formaldehyde vapor to which employees are exposed. Formalin. Formalin refers to stabilized aqueous solutions which contain 37 to 40% formaldehyde and which are commercially available. For laboratory use this solution is normally diluted in buffered water to approximately 4%. Formaldehyde gas is combustible and may form explosive mixtures in air when the concentration exceeds 7%, a concentration which may be exceeded in the event of a fire in a room in which formalin is stored. Formalin should therefore be stored in a wellventilated area away from oxidizing agents. Diethyl ether. Although most parasitology laboratories have substituted ethyl acetate for diethyl ether in their concentration procedure, caution is still necessary. The reagent should be stored in an explosion-proof cabinet and used in a chemical fume hood, and all areas where it is used must be free of sources of ignition. Disposal of small quantities of ether is best handled by evaporation in a chemical fume hood. Larger quantities should be transported in durable, explosion-proof containers to special facilities and disposed of in accordance with federal or state requirements. Liquid nitrogen. Liquid nitrogen is used mainly for the storage of protozoan cultures, and the handling of this material requires certain precautions. The storage container should be in a wellventilated room, and impervious gowns, face masks, protective glasses, and insulated gloves should be worn when opening the container. Care should be taken when pouring liquid nitrogen from one container to another, as extensive contact through soaked clothing can cause severe burns. Picric acid. Picric acid is normally stable in solution, as used in a routine laboratory in the iron hematoxylin stain; however, if allowed to evaporate to dryness, it is highly explosive. It may cause irritation or allergic eruptions on contact with skin. CURRENT
AND WORLDWIDE BIOSAFETY INFORMATION Much of laboratory safety is directly related to awareness of new information with respect to newly emerging problems and new products and solutions. Often the questions at hand in one laboratory are already being discussed in others. Fortunately, new information can be sought and obtained on a rapid and worldwide basis through the internet, e-mail, and the World Wide Web. This information is readily and inexpensively available via the computer and a modem and appropriate connecting software. A short list of infor-
16
JAMISON
ET AL.
mation sites and discussion newsgroups is as follows: American Society for Microbiology http://www .asmusa.org CDC http://www.cdc.gov World Health Organization http://www.who.ch Health Canada hcp://hpbl.hwc:ca Biosafety line e-mail message: sub biosafety firstname lastname to
[email protected] Promed e-mail message: sub promed firstname lastname to
[email protected] CONCLUSION It is clear that while laboratories may differ in specifics with respect to hazards and safety requirements, there are some elements of a safety program that are common to all of them. Management must make employees aware of the requirements and practices inherent in safe laboratory practice. Each laboratory and laboratory section should have a comprehensive safety manual with sections indexed for easy access. Laboratory employees must be required to read and familiarize themselves with the safety manual, and compliance with this requirement should be recorded. Further, management has an obligation to provide a safe workplace for the employee. Facilities and equipment suitable for containment of biohazards, disinfection of laboratory accident sites, and sterilization of pathogens must be readily available. Personal protective equipment should be provided, and usage of equipment appropriate for each task should be required. Employees have an obligation to themselves, to their employer, and to the general public to be conscious of safety and of biohazards. They should conduct themselves at all times in a manner most conducive to their own safety and that of their colleagues and the public. Employees must be safety conscious, heed the regulations, and take upon themselves the responsibility for their own safety. This implies that each employee joins management in promulgating safety consciousness and participates in development of safe practices and procedures in the laboratory. The names and telephone numbers of safety ofl%ers and fire wardens should be listed. It is management’s responsibility to inculcate the need for safety consciousness in all employees, since safety, particularly microbiological safety, is not only an individual concern but a community imperative. REFERENCES 1. Allen, B. W., and J. H. Darrell. 1981. Extrapulmonary tuberculosis: a potential source of laboratory-acquired infection. J. Clin. Pathol. 17~242-243. 2. Allen, B. W., and J. H. Darrell. 1983. Contamination of specimen container surfaces during sputum collection. J. Clin. Pathol. 361479-481.
CUMITECH
29
3. Almeida, J. D. 1980. Practical aspects of diagnostic electron microscopy. Yale J. Biol. Med. 53:5-18. 4. American Public Health Association. 1989. In E. H. Lennette and N. J. Schmidt (ed.), Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 6th ed. 5. Andre, F. E. 1989. Summary of safety and efficacy data on a yeast-derived hepatitis B vaccine. Am. J. Med. 87(Suppl. 3): 14S-20s. 6. Ashdown, L., and J. Cassidy. 1991. Successive Salmonella give and Salmonella typhi infections, laboratory-acquired. Pathology 23~233-234. 7. Batchelor, B. I., R. J. Brindle, G. F. Gilks, and J. B. Selkon. 1992. Biochemical mis-identification of Brucella melitensis and subsequent laboratory acquired infections. J. Hosp. Infect. 22~158-162. 8. Blaser, M. J., F. W. Hickman, J. J. Farmer III, D. J. Brenner, A. Balows, and R. A. Feldman. 1980. Salmonella typhi: the laboratory as a reservoir of infection, J. Infect. Dis. 142:934 -938. 9. Blaser, M. J., and J. P. Lofgren. 1981. Fatal salmonellosis originating in a clinical microbiology laboratory. J. Clin. Microbial. 13:855- 858. 10. Burnens, A. P., R. Zbinden, L. Kaempf, I. Heinzer, and J. Nicolet. 1993. A case of laboratory acquired infection with Escherichia coli 0157:H7. Zentralbl. Bakteriol. 279~512-517. 11. Centers for Disease Control. 1988. Update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus and other bloodborne pathogens in healthcare settings. Morbid. Mortal. Weekly Rep. 37~377-382. 12. Centers for Disease Control. 1992. Seroconversion to simian immunodeficiency virus in two laboratory workers. Morbid. Mortal. Weekly Rep. 41~678-681. 13. Centers for Disease Control and Prevention. 1993. Biosafety in Microbiological and Biomedical Laboratories, 3rd ed. DHHS publication no. (CDC)938395. U.S. Department of Health and Human Services, Atlanta. 14. Centers for Disease Control and Prevention. 1994. Laboratory management of agents associated with hantavirus pulmonary syndrome: interim biosafety guidelines. Morbid. Mortal. Weekly Rep. 43~1-7. 15. Centers for Disease Control and Prevention. 1994. Bolivian hemorrhagic fever-El Beni Department, Bolivia. Morbid. Mortal. Weekly Rep. 43~943-946. 16. Chan, S. K., D. Vesley, L. M. Brousseau, and J. H. Vincent. 1994. Evaluation of single-use masks and respirators for protection of health care workers against mycobacterial aerosols. Am. J. Infect. Control 22:65-74. 17. Charous, B. L., R. G. Hamilton, and J. W. Yunginger. 1994. Occupational latex exposure: characteristics of contact and systemic reactions in 47 workers. J. Allergy Clin. Immunol. 94~12-18. 18. Doane, F .W., and N. Anderson. 1987. Electron Microscopy in Diagnostic Virology. A Practical Guide and Atlas. Cambridge University Press, Cambridge. 19. Fedson, D. S. 1993. Immunizations for health-care workers and patients in hospitals, p. 214-294. In R. P. Wenzel (ed.), Prevention and Control of Nosocomial Infections, 2nd ed. The Williams & Wilkins Co., Baltimore. 20. Georghiou, P. R., and E. J. Young. 1991. Prolonged incubation in brucellosis. Lancet 337:1543. 21. Greuner, E., E. Bernasconi, R. L. Galeazzi, D. Buhl, R. Heinzle, and D. Nadal. 1994. Brucellosis: an occupational hazard for medical laboratory personnel. Report of 5 cases. Infection 22~33-36. 22. Grist, N. R., and J. A. N. Emslie. 1994. Association of clinical pathologists’ surveys of infection in British clinical laborato&es. J. clin. Pathoi. 47~391-394. 23. Guibourdenche, M., J.-P. Darchis, A. Boisivon, E. Collatz, and J.-Y. Riou. 1994. Enzyme electrophoresis, sero- and subtyping, and outer membrane protein characterization of two Neisseria meningitidis strains involved in laboratory-acquired infections. J. Clin. Microbial. 32:701-704.
CUMITECH
29
LABORATORY
Canada. 1990. Laboratory Biosafety Guidelines. 24. Health Health Canada and Medical Research Council of Canada. B. L., and D. D. Juranek. 1993. Laboratory25. Herwaldt, acquired malaria, leishmaniasis, trypanosomiasis and toxoplasmosis. Am. J. Trap. Med. Hyg. 48:313-323. Air Transport Association. 1995. Dangerous 26. International Goods Training Program, 19th ed. International Air Transport Association, Montreal, Canada. H. D. (ed.). 1994. Clinical Microbiology Procedures 27. Isenberg, Handbook, Supplement 1. American Society for Microbiology, Washington, D.C. J. A. (ed.). 1990. Histological and Histochemical 28. Kiernam, Methods. Theory and Practice, 2nd ed. Peigamon Press, New York. D. M., M. Kit-win, K. Cresci, and T. Sing. 1994. 29. Korniewicz, Barrier protection with examination gloves: double versus single. Am. J. Infect. Control 22~12-15. V. H., G. J. Harper, and A. E. Wright. 1983. An 30. Lath, assessment of some hazards associated with the collection of venous blood. J. Hosp. Infect. 4:57-63. 1967. A fluorescent complex 31. Le Recq, J. S., and C. Paoletti. between ethidium bromide and nucleic acids. J. Mol. Biol. 27:87-106. G. A., R. Brindle, P. N. Sockett, J. Solera, P. 32. Luzzi, Klenerman, and D. A. Warrell. 1993. Brucellosis: imported and laboratory acquired cases and an overview of treatment trials. Trans. R. Sot. Trop. Med. Hyg. 87:138-141. Research Council. 1989. Biosafety in the Labora33. National tory. Prudent Practices for the Handling and Disposal of Infectious Materials. National Academy Press, Washington, D.C. M. D., M. Frederickson, N. L. Good, and S. A. 34. Nettleman, Hunter. 1994. Tuberculosis control strategies: the cost of particulate respirators. Ann. Intern. Med. 122:37-40. P. J. M. 1991. Accidental infection of laboratory 35. Openshaw, worker with recombinant vaccinia virus. Lancet 338:459. G. B. 1986. Human factors in microbiological 36. Phillips, laboratory accidents, p. 43-48. In B. M. Miller, D. H. M. Groschel, J. H. Richardson, D. Vesley, J. R. Songer, R. D. Housewright, and W. E. Barkley (ed.), Laboratory Safety: Principles and Practice. American Society for Microbiology, Washington, D.C. acquired infections: summary 37. Pike, R. M. 1976. Laboratory and analysis of 3,921 cases. Health Lab. Sci. 13:105-114. with 38. Pike, R. M. 1978. Past and present hazards of working infectious agents. Arch. Pathol. Lab. Med. 102:333-336. acquired infections: incidence, 39. Pike, R. M. 1979. Laboratory fatalities, cases and prevention. Annu. Rev. Microbial. 33:4166. M. C., P. M. Tierno, P. Ottuso, A. B. Klemes, 40. Raviglione, and M. Davidson. 1990. Group G streptococcal meningitis and sepsis in a patient with AIDS. Diagn. Microbial. Infect. Dis. 13:261-264. M. C., M. LeBras, and P. Vincendeau. 1993. 41. Receveur, Laboratory-acquired Gambian trypanosomiasis. N. Engl. J. Med. 329:209-210. Systems. 1993. AMPLICOR Chlamydia 42. Roche Diagnostic trachomatis Test Package Insert. Roche Diagnostic Systems, Nutley, N.J. and M. A. Noble. 1990. Hepatitis 43. Roscoe, D. L., G. Gibson, B and HIV infections in dental professionals in British Columbia. CDWR 16:175-176.
SAFETY
IN CLINICAL
MICROBIOLOGY
17
W. A. 1993. Disinfection, sterilization and waste 44. Rutala, disposal, p. 460-496. In R. P. Wenzel (ed.), Prevention and Control of Nosocomial Infections, 2nd ed. The Williams & Wilkins Co., Baltimore. W. F., III, J. B. Turchik, R. E. Westlake, Jr., G. C. 45. Schlech, Klein, J. D. Band, and R. E. Weaver. 1981. Laboratoryacquired infection with Pseudomonas pseudomallei (melioidosis). N. Engl. J. Med. 303:519-521. as a contact 46. Seal, D. V., and J. Hay. 1993. The microbiologist lens wearer. J. Med. Microbial. 39:39-40. V. K., B. Kurnar, B. D. Radotra, and S. Kumar. 47. Sharma, 1990. Cutaneous inoculation in tuberculosis in laboratory personnel. Int. J. Derrnatol. 29:293-294. C. H., C. J. Brandt, P. J. Middleton, and J. A. 48. Sherlock, Smith. 1989. Cumitech 26, Laboratory Diagnosis of Viral Infections Producing Enteritis. Coordinating ed., J. A. Smith. American Society for Microbiology, Washington, D.C. of Japanese Virologists. 1993. Guidelines for bio49. Society safety in virus research. Virusu 43:199-232. E. H. 1964. Alcohol as a surgical disinfectant. 50. Spaulding, Assoc. Operating Room Nurses J. 2~67-71. Australia. 1991. Safety in Laboratories. Part 3. 51. Standards Microbiology, 3rd ed. AS 2243.3. Standards Australia, Standards House, North Sidney, Australia. J., C. M. Lewis, J. Colville, M. Zervos, and J. 52. Staszkiewicz, Band. 1991. Outbreak of Brucella melitensis among microbiology laboratory workers in a community hospital. J. Clin. Microbial. 29:287-290. S. E., and R. M. Pike. 1951. Survey of laboratory 53. Sulkin, acquired infections. Am. J. Public Health 41:769-781. F. C., J. T. Crawford, R. E. Huebner, L. J. Geiter, 54. Tenover, C. R. Horsburgh, Jr., and R. C. Good. 1993. The resurgence of tuberculosis. Is your laboratory ready? J. Clin. Microbial. 31:767-770. Committee on Dangerous Pathogens. 1984. 55. U.K. Advisory Categorization of Pathogens According to Hazard and Categories of Containment. Her Majesty’s Stationery Office, London. C. H., B. J. Erasmus, R. Swanepoel, and 56. Van der Meyden, 0. W. Prozesky. 1992. Encephalitis and chorioretinitis associated with neurotrophic African horsesickness virus infection in laboratory workers. Part 1. Clinical and neurological observations. S. Afr. Med. J. 81:451--454. and P. Isoard. 1993. 57. Vidal, D. R., J. C. Paucod, F. Thibault, Biosecurite au laboratoire. Risque biologique, normalisation et pratique. Ann. Pharm. Fr. 51:154-166. K. 1993. Laboratory improvement: the dirty dozen 58. Voldish, for OSHA. N.J. Med. 90:602-603. R. G., and W. Noe. 1993. Mammalian cell culture. 59. Werner, Part III. Safety and future aspects. Arzneimittelforschung 43:1388-1390. J. E. 1983. Warning on a new potential for 60. Williams, laboratory-acquired infections as a result of the new nomenclature for the plague bacillus. Bull. W. H. 0. 61:545-546. J. W., C. E. Smith, and 0. A. Plunkett. 1953. 61. Wilson, Primary cutaneous coccidioidomycosis; the criteria for diagnosis and a report of a case. Calif Med. 79:233-239. Health Organization. 1983. Laboratory Biosafety 62. World Manual. World Health Organization, Geneva.
