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? التعرف على المخاطر الحيوية وطرق التحكم Biohazard Recognition and Control Dr.Nagwa Aref Dr.Nagwa Aref Dr.Nagwa Aref Dr.Nagwa Aref بعض المخاطر أجريت على ميكروب brucellosisدراسة سنة 1941في الواليات األمريكية تم حصر 74حالة إصابة بمرض الحمى المتموجة عينات المرضى عند استالمها أو اإلهمال وعدم أخذ الحيطة والحذر عند التعامل مع مزارع تلك العينات إما عن طريق استنشاق غبار ملوث بميكروب الحمى المتموجة من تلك العينات أو المالمسة أو ضعف التقنية المتاحة في التعامل مع مثل هذا الميكروب الخطير والسريع االنتشار سنة 1949ذكرت وجود 222حالة إصابة بمرض فيروسي كانت بسبب معامل تحاليل وأبحاث منها 21حالة كانت قاتلة وكان معظمها بسبب عدم التعامل السليم مع األنسجة أو الحيوانات المصابة حاالت مرض السل، من تلك ) %20 , (Venezuelan encephalitisالحمى المتموجة ،والتيفويد والتهابات تليف الكبد والدماغ وباألخص مرض التهاب الدماغ الفنزويلي الحاالت فيها طريقة التعرض للميكروب معروفة وأدت إلى اإلصابة أما الباقي فيعتقد أن المسبب كان التعرض باالستنشاق المباشر بحكم تعامل المصابين مع عينات و هذه المجموعة تضم عدة )(Arbovirusesالمواد المعدية المسببة لتلك األمراض وفي سنة 1967تم الكشف عن 428حالة إصابة بفيروسات بسبب التعامل مع مجموعة فيروسات ممرضة تنتقل لإلنسان عن طريق الحشرات – – – (encephalitis) ،وفيروسات التهاب الدماغ بأنواعها المختلفة ) (yellow feverتشمل فيروسات الحمى الصفراء (arthropod-borne viruses معظمها كان بسبب التعرض لبخار أو غبار أو بقايا صغيرة جدا ً متطايرة من عينات قادمة للمعامل للتشخيص اإلصابات الفطرية أيضا ً ذكرت في عدة دراسات وكانت معظمها بسبب أخطاء بشرية من العاملين بالمختبرات البحثية منها تناثر قطرات من مزارع فطرية على أو إصابات الجهاز التنفسي بسبب تناثر العفن الطبي )(Sporothrix schenckiiالعيون وخدش وحقن مواد ملوثة عبر الجلد بفطريات ممرضة مثل ) (Histoplasma capsulatumو )(Blastomyces dermatitidis – – (Neisseriaالالمباالة كانت السبب وراء إصابة خمسة أشخاص بمرض التهاب السحايا وذلك عندما حُضر مستحلب من الميكروب وكان ذلك قبل سبعة أيام من ظهور أعراض المرض ) (safety cabinetخارج حجرة العزل )meningitidis ل(Laboratory-acquired parasitic infectionsواإلصابات المعملية الطفيلية م تكن استثناء من ذلك وخاصة بالدول النامية، – – – – وفي دراسة أخرى تمت مناقشة 164حالة إصابة طفيلية للعاملين بمجال المختبرات الطبية من حيث األسباب التي أدت إلى ذلك .إصابات شملت أمراض طفيليات Dr.Nagwa Aref – Dr.Nagwa Aref Dr.Nagwa Aref RECOMBINANT DNA AND GENETIC MANIPULATION USE OF MAMMALIAN CELLS IN CULTURE Dr.Nagwa Aref RISK LEVELS ASSOCIATED WITH THE USE OF LABORATORY ANIMALS CRITERIA FOR CLASSIFICATION OF BIOLOGICAL AGENTS BY RISK GROUP CRITERIA FOR CLASSIFICATION OF BIOLOGICAL AGENTS BY RISK GROUP Risk Group 1 Risk Group 2 Risk Group 3 Risk Group 4 low individual risk, moderate individual risk, high individual risk high individual risk limited community risk limited community risk low community risk high community risk Dr.Nagwa Aref Risk Group 2 Laboratory exposures rarely cause infection leading to serious disease Effective treatment and preventive measures are available The risk of spread is limited. Risk Group 3 A pathogen that usually causes serious human or animal disease, or which can result in serious economic consequences Does not ordinarily spread by casual contact from one individual to another can be treated by antimicrobial or Dr.Nagwa Aref antiparasitic agents Risk Group 2 A pathogen that usually causes serious human or animal disease, can result in serious economic consequences but does not ordinarily spread by casual contact from one individual to another, or that can be treated by antimicrobial or antiparasitic agents. BIOLOGICAL AGENTS BY RISK GROUP Chlamydia Mycoplasma Bacteria Fungi Parasites Viruses parasites have caused laboratory infections by ingestion, skin or mucosal penetration or accidental injection. Agents listed in this group may be present in blood, CSF, central nervous system and other tissues, and infected arthropods, depending on the agent and the stage of infection. Dr.Nagwa Aref Bacteria Risk Group 2 Actinobacillus - all species Leptospira interrogans - all serovars Actinomyces pyogenes (C. pyogenes) Listeria monocytogenes Bacillus cereus Mycobacteria - all species (except M. tuberculosis, and M. bovis Bartonella bacilliformis, B. henselae, B. quintana, B. elizabethae Mycoplasma pneumoniae, M. hominis Neisseria gonorrhoeae, N. Bordetella pertussis, B. parapertussis and B. meningitidis bronchiseptica Nocardia asteroides, N. brasiliensis Borrelia recurrentis and B. burgdorferi Pasteurella, all species (except P. multocida type B in Level 3) Campylobacter spp. (C. coli, C . fetus, C. jejuni) Pseudomonas aeruginosa Chlamydia pneumoniae, C. psittaci (non-avian Salmonella enterica (S. choleraesuis) strians), C. trachomatis, Salmonella enterica serovar arizonae (Arizona hinshawii) Clostridium botulinum, Cl. chauvoei, Cl. difficile, Cl. Salmonella enterica ser. gallinarum-pullorum (S. gallinarum- haemolyticum, pullorum) Cl. histolyticum, Cl. novyi, Cl. perfringens, Cl. Salmonella enterica ser. meleagridis (S. meleagridis) septicum, Salmonella enterica ser. paratyphi B (S. paratyphi B) Cl. sordellii, Cl. tetani (Schottmulleri) Corynebacterium diphtheriae, C. haemolyticum, Salmonella enterica ser. typhi (S. typhi) C. pseudotuberculosis, C. pyogenes (A. pyogenes) Salmonella enterica ser. typhimurium (S. typhimurium) Edwardsiella tarda Shigella boydii, S. dysenteriae, S. flexneri, S. sonnei Erysipelothrix rusiopathae (insidiosa) Staphylococcus aureus Escherichia coli Streptobacillus moniliformis enterotoxigenic/invasive/hemorrhagic strains Streptococcus spp. (Lancefield Groups A, B, C, D, G) Francisella tularensis Type B, (biovar palaearctica), F. novocida Fusobacterium necrophorum Haemophilus influenzae, H. ducreyi Helicobacter pylori (non-BCG strain), which are in Risk Group 3) Treponema carateum, T. pallidum (including pertenue), T. vincentii Ureaplasma urealyticum Vibrio cholerae (incl. El Tor), V. parahaemolyticus, V. vulnificus Dr.Nagwa ArefYersinia enterocolitica, Y. pseudotuberculosis Virus Risk Group 2 – Adenoviridae Adenoviruses, all serotypes Arenaviridae – Lymphocytic choriomeningitis virus (laboratoryadapted strains) – Tacaribe virus complex: Tamiami, Tacaribe, Pichinde Bunyaviridae* – – – – – – – – – – Genus Bunyavirus – Bunyamwera and related viruses – California encephalitis group, including LaCrosse, Lumbo and snowshoe hare Genus Phlebovirus – – – All species except Rift Valley fever virus Coronaviridae – Human coronavirus, all strains – Transmissible gastroenteritis virus of swine – emagglutinating encephalomyelitis virus of swine Genus Lymphocryptovirus: Epstein Barr Virus (HHV 4) and EB-like isolates – – – – Mouse hepatitis virus Bovine coronavirus Feline infectious peritonitis virus Avian infectious bronchitis virus Canine, Rat and Rabbit coronaviruses Flaviviridae* Yellow fever virus (17D vaccine strain) Dengue virus (serotypes 1,2,3,4) Kunjin virus Hepadnaviridae Hepatitis B virus, includes Delta agen Herpesviridae Alphaherpesvirinae Genus Simplexvirus: all isolates, including HHV 1 and HHV 2, except Herpes B virus which is in Risk Group 4 Genus Varicellovirus: all isolates, including varicella/zoster virus (HHV 3) and pseudorabies virus (see Table 1) Betaherpesvirinae Genus Cytomegalovirus: all isolates including CMV (HHV 5) Genus Muromegalovirus: all isolates Gammaherpesvirinae Genus Paramyxovirus: all isolates Genus Pneumovirus: all isolates – Genus Morbillivirus: all isolates (except Rinderpest-see Table 1) Parvoviridae Genus Parvovirus: all isolates Picornaviridae – Genus Rhadinovirus: all isolates (except H. ateles and H. saimiri, see Risk Group 3) – Genus Thetalymphocryptovirus: all isolates Unassigned Herpesviruses: includes HHV 6 (human á-lymphotrophic virus), HHV 7, HHV 8, etc. Orthomyxoviridae – Genus Aphthovirus - See Table 1 – Genus Influenzavirus: – Genus Cardiovirus - all isolates – Influenza virus type A, all isolates – Influenza virus type B, all isolates – – Influenza virus type C, all isolates Papovaviridae Genus Enterovirus - all isolates (see Table 1 for restrictions) – Genus Papillomavirus: all isolates – Genus Hepatovirus - all isolates (Hepatitis A) – Genus Polyomavirus: all isolates Paramyxoviridae Dr.Nagwa Aref – Genus Rhinovirus - all isolates BIOLOGICAL AGENTS BY RISK GROUP Bacillus anthracis Brucella - all species Burkolderia (Pseudomonas) mallei; B. pseudomallei Chlamydia psittaci - avian strains only Coxiella burnetii Francisella tularensis, type A (biovar tularensis) Mycobacterium tuberculosis; M. bovis (non-BCG strains) Chlamydia Mycoplasma Pasteurella multocida, type B Bacteria Fungi Bunyaviridae Unclassified Bunyavirus Hantaan, Korean haemorrhagic fever and epidemic nephrosis viruses including virus responsible for Hantavirus pulmonary syndrome) Rift Valley fever virus Flaviviridae* Yellow fever virus (Wild type) Japanese encephalitis virus Murray Valley encephalitis Viruses Human immunodeficiency viruses (HIV - all isolates Dr.Nagwa Aref BIOLOGICAL AGENTS BY RISK GROUP Arenaviridae Lassa, Junin, Machupo viruses, Sabia, Guanarito Bunyaviridae* Genus Nairovirus Crimean-Congo hemorrhagic fever Filoviridae Marburg virus Ebola virus Flaviviridae* Viruses Tick-borne encephalitis complex, including Russian Spring-Summer Encephalitis Kyasanur forest virus Omsk hemorrhagic fever virus Herpesviridae Alphaherpesvirinae Genus Simplexvirus: Herpes B virus (Monkey virus) Poxviridae Genus Orthopoxvirinae Variola Monkeypox Dr.Nagwa Aref • Pathogenicity of material – disease incidence and severity • Routes of Transmission – parenteral, airborne or ingestion • Agent Stability – ease of decontamination • Infectious Dose – LD50 • Concentration – infectious organisms/vol. & working volume • Origin of material - Wild Type, exotic, primary cells • Availability of effective prophylaxis – Hep. B vaccine • Medical surveillance – exposure management • Skill level of staff Dr.Nagwa Aref Risk Assessment • Risk of Activity – same agent can have different containment levels at different stages of protocol: – – – – – Procedures that produce aerosols have higher risk Procedures using needles or other sharps have higher risk Handling blood, serum or tissue samples may have lower risk Purified cultures or cell concentrates may have higher risk Larger volumes (10 L) have higher risk Dr.Nagwa Aref Primary Containment • • • • Lab practices – standard lab practice, limited access, biohazard warning sign, sharps/needle precautions, SOPs for decon, waste, medicals. Safety equipment – biosafety cabinets (BSC), sharps containers, sealed rotors. Personal protective equipment (PPE) – lab coat, gloves, goggles. Host-vector for rDNA Dr.Nagwa Aref Dr.Nagwa Aref Dr.Nagwa Aref Four Levels of Biosafety • BSL 1: Material not known to consistently cause disease in healthy adults. • BSL 2: Associated with human disease. Hazard is from percutaneous injury, ingestion, or mucous membrane exposure. • BSL 3: Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences. • BSL 4: Dangerous/exotic agents which pose a high risk of lifethreatening disease, aerosol-transmitted lab infections or related agents with unknown risk of transmission. Dr.Nagwa Aref Human Blood, Tissue and Fluid • Occupational Exposure to Bloodborne Pathogens • Occupational Safety & Health Administration (OSHA) 29 CFR 1910.1030 • • • • • • • Use BSL 2 work practices and procedures. Additional requirements for HIV work. Everyone needs to be offered the Hepatitis B vaccine. Develop specific exposure plan SOPs. Specific training is required. Review needle/syringe use and replace with “safe” devices. Exposure incidents must be followed up. Dr.Nagwa Aref Aerosol Precautions • Use BSC for all procedures that may generate aerosols. • Use centrifuges with biosafety covers. • Do not use a syringe for mixing infectious fluids. • Cultures, tissues, specimens of body fluids, etc., are placed in a container with a cover that prevents leakage during collection, handling, processing, storage, transport or shipping. Dr.Nagwa Aref Select Agents • Possession, use and transfer of specific biological agents requires registration • “Restricted Persons” are not allowed to have access to these agents. • High security and containment must be maintained. • Web site: http://www.cdc.gov/od/o hs/lrsat.htm Dr.Nagwa Aref • Control access to areas where biological agents or toxins are used and stored. • Keep biological agents and toxins in locked containers. • Know who is in the lab. • Know what materials are being brought into the lab. • Know what materials are being removed from the lab. • Have a protocol for reporting incidents. • Have an emergency plan. Dr.Nagwa Aref Waste Disposal • “Red bag” or “Regulated Medical Waste”: – All mammalian cells or anything that came in contact with mammalian cells – All BSL 2 material or anything that came in contact with BSL 2 material – All needles/syringes regardless of use • No need to autoclave this waste prior to disposal in EH&S red bag/box (material is incinerated). Dr.Nagwa Aref Dr.Nagwa Aref نظام فرز المخلفات ()System of Segregation .1المخلفات الطبية المعدية .2 المخلفات الطبية المعدية األخرى مثل األعضاء واألنسجة البشرية .3المخلفات الحادة .4المخلفات الصيدالنية والكيماوية .5المخلفات المشعة .6المخلفات الغير الطبية Dr.Nagwa Aref العالمـــــــات الدولـــــــــية المخلفات البيولوجية والطبية المعدية المخلفات الطبية المشعة مخلفات سريعة االشتعال المخلفات الكيماوية شديدة التفاعل مع الماء المخلفات الطبية ذات األضرار البيئية مخلفات أدوية سامة مخلفات كيماوية حارقة Dr.Nagwa Aref • OSHA Bloodborne Pathogens • • • • • • • • • • • http://www.osha.gov/SLTC/bloodbornepathogens/index.html CDC Select Agents http://www.cdc.gov/od/ohs/lrsat.htm NIH Guidelines for Research Involving Recombinant DNA Molecules http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html DOT/CDC Shipping http://www.cdc.gov/od/ohs/biosfty/shipregs.htm CDC Import Permits http://www.cdc.gov/od/ohs/biosfty/imprtper.htm USDA/APHIS Permits http://www.aphis.usda.gov/vs/ncie/ Dr.Nagwa Aref • CDC: a permit is required to import etiologic agents of human disease and any materials, including live animals or insects, that may contain them. Unsterilized specimens of human and animal tissues (such as blood, body discharges, fluids, excretions or similar material) containing an infectious or etiologic agent require a permit in order to be imported. • APHIS: a USDA veterinary permit is needed for materials derived from animals or exposed to animal-source materials. Dr.Nagwa Aref • Develop and practice plans for: • Spills: large spills, spills inside BSC – Accidental exposures: needlesticks, eye/mucous membrane splash, breathing aerosols – Power/Utility failures: BSC, freezers, ventilation, lights, water – Fires – Medical emergencies Dr.Nagwa Aref Resources • CDC Biosafety in Microbiological and Biomedical Laboratories • http://www.cdc.gov/od/ ohs/biosfty/bmbl4/bmb l4toc.htm • ABSA Risk Groups • http://www.absa.org/ris kgroups/index.htm • Canadian MSDSs • http://www.hcsc.gc.ca/pphbdgspsp/msdsftss/index.html • Environmental Health & Safety – Lab Safety • http://www.ehs.sunysb. edu or 2-9672 Dr.Nagwa Aref