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Wednesday, May 23, 2012

Sunday, May 20, 2012

Lectures in Clinical Microbiology: Diagnoses of Infectious Diseases

Lectures in Clinical Microbiology: Diagnoses of Infectious Diseases: Diagnosis of infectious diseases in clinical microbiological laboratory is attempted through following methods: - Conventional Methods La...

Diagnoses of Infectious Diseases

Diagnosis of infectious diseases in clinical microbiological laboratory is attempted through following methods: - Conventional Methods Laboratory tests may identify organisms either directly microscopically by stains, growing organisms by cultural techniques and/or detecting their antigens or indirectly by identifying antibodies to the organism. -Molecular Methods This techniques Identify organisms by detecting their DNA or RNA by specific technologies. Conventional technologies for identifying microorganisms usually involve good sampling techniques to assure accurate laboratory diagnosis. Conventional Methods for Diagnosis of Infectious Diseases Infectious diseases are common diseases all over the world. A recent World Health Organization report indicated that infectious diseases are now the world’s biggest killer of children and young adults. Infectious diseases in non-industrialized countries caused 45% in all and 63% of death in early childhood. Causes of Infectious Diseases: The microbial causes of human diseases are classified into theses groups: 1- Bacteria 2- Viruses 3- Fungi 4- Protoza 5- Chlamydiae 6- Rickettsiae 7- Mycoplasmas. Infection may be endogenous or exogenous. 1- Endogenous infections: the microorganism (usually a bacterium) is a component of the patient's endogenous flora. Endogenous infections can occur when the microorganism is aspirated from the upper to the lower respiratory tract or when it penetrates the skin or mucosal barrier as a result of trauma or surgery. 2- Exogenous infections: the microorganism is acquired from the environment (e.g., from soil or water) or from another person or an animal. The ability to control such microbial infections is largely dependent on the ability to detect these etiological agents in the clinical microbiology laboratory (Millar et al., 2003) Diagnoses of Infectious Diseases Diagnosis of infectious diseases in clinical microbiological laboratory is attempted through following methods: - Conventional Methods Laboratory tests may identify organisms either directly microscopically by stains, growing organisms by cultural techniques and/or detecting their antigens or indirectly by identifying antibodies to the organism. -Molecular Methods This techniques Identify organisms by detecting their DNA or RNA by specific technologies. Conventional technologies for identifying microorganisms usually involve good sampling techniques to assure accurate laboratory diagnosis. Figure (1): Microbiology laboratory techniques for diagnosis of infectious diseases. A-Proper Sampling in Clinical Microbiology Laboratory Specimens selected for microbiologic examination should reflect the disease process and collected in sufficient quantity to allow complete microbiologic examination. The number of microorganisms per milliliter of a body fluid or per gram of tissue is highly variable, ranging from less than 1 to 108 or 1010 colony-forming units (CFU). Collection of good quality specimens depends on: -The optimal time of specimen collection. -The correct type of specimen -Well collected specimens with minimum contamination from normal flora of the patient or the person collecting the specimen. -Adequate amounts of each specimens and appropriate number of specimens -Clearly labeled safe specimens Optimal Time of Collection of Specimens The proper time of sampling in clinical microbiology play a crucial role in proper laboratory diagnosis. There are several examples for such proper time. If possible, specimens should be collected before the administration of antibiotics. Above all, close communication between the clinician and the microbiologist is essential to ensure that appropriate specimens are selected and collected and that they are appropriate -Blood cultures and blood films for malarial parasites are best collected just as the patient’s temperature starts to rise, however, when infective endocarditis is suspected, three blood culture sets collected with 24 hour irrespective of patient, s temperature. - Specimens for virus isolation are most likely to give positive results when collected during the most acute stages of the disease -Serology is satisfactory when four fold or greater rising antibody titer is demonstrated in pained sera. The 1st serum sample as early as possible in the disease course. Second in the convalescent stage. Correct Types of Specimens Examples: Bacterial meningitis-------blood cultures, CSF culture Suspected gonorrhea -------cervical, urethral and rectal swab should be collected rather than high regional swabs. Well Collected Specimens with Minimum Contamination from the Normal Flora The good quality of microbiological sample is a need for accurate laboratory diagnosis. The main problem is mixing of samples with normal flora normally resident beside infected tissue. Skin and mucous membranes have a large and diverse endogenou flora; so every effort must be made to minimize specimen contamination during collection. Contamination may be avoided by various means: - The skin can be disinfected before aspirating or incising a lesion. - The contaminated area may be bypassed altogether. Examples of such approaches are trans tracheal puncture with aspiration of lower respiratory secretions or supra pubic bladder puncture with aspiration of urine. It is often impossible to collect an uncontaminated specimen, and decontamination procedures, cultures on selective media, or quantitative cultures must be used. Specimens collected by invasive techniques, particularly those obtained intra operatively, require special attention. Enough tissue must be obtained for both histopathologic and microbiologic examination. Adequate Amounts of Appropriate Number of Specimens The volume of blood for culture from adult -5-10 ml per bottle and in children and neonates 1-5ml per bottle. -Collection of early morning sputum specimens, and collection of adequate amount of early morning urine specimen for 3 successive days is required for the isolation of Mycobacterium tuberculosis (TB). -Patients with diarrhea ---at least 2 specimens of stool are collected for culture of Salmonellae spp. or Shigella spp. -Serological investigations usually require paired sera. Clearly Labeled and Safe Specimens Specimens for microbiological investigations should be placed in leak – proof containers, and each container should be enclosed in plastic bag. The hazards to staff handling leaking container s include acquiring enteric infection from feces, TB from sputum of an open case of pulmonary TB and viruses s such as HCV, HBV, HIV, from leaking blood. B-Transport of specimens to the laboratory The specimen must be transported rapidly, in the correct medium, and in conditions that limit growth of any potentially contaminating normal flora. For accurate quantification of the pathogen, additional pathogen growth must be prevented; specimens should be transported to the laboratory immediately or, if transport is delayed, refrigerated (in most cases). Certain cultures have special considerations. Many pathogenic organisms don’t survive for long in clinical specimens kept at room temperature. Examples include Gonococci, Haemophilus, Bacteroides, anaerobic cocci and most viruses. On the other hand, some organisms contaminating specimens from the normal flora such as Coliform and Coagulase negative Staphylococci, may rapidly grow in specimen kept at room temp. -Urine or sputum specimens should reach the laboratory within 2hours of collection when even possible. If delay is expected immediately inoculated into transport media. -Transport media used: Stuart’s transport media ----- for pus or swabs for bacterial culture when delays in transport.>1/2hour or when Neisseria infections are suspected. However the inoculated transport media should be sent to the laboratory within 4hours. Cerebrospinal fluid(CSF) not refrigerated since other wise Meningococci may rapidly die. Viral transport media is necessary for virus isolation, and also for Chlamydia isolation. Specimens for virus isolation are kept at –70ÂșC till time of transferring the appropriate cell line which support growth of the possible virus or Chlamydia. Figure (2) Laboratory procedures used in confirming a clinical diagnosis of infectious disease with a bacterial etiology. C- Conventional laboratory Methods for Diagnosis of Infectious Diseases 1-Direct method: They are Laboratory tests may identify organisms directly (eg, visually, using a microscope, growing the organism in culture) I- Microscopy Microscopy can be done quickly, but accuracy depends on the experience of the microscopist and quality of equipment. Regulations often limit physicians' use of microscopy for diagnostic purposes outside a certified laboratory (Siqueira, J. Fet al., 2005). Most specimens are treated with stains that color pathogens, causing them to stand out from the background, although wet mounts of unstained samples can be used to detect fungi, parasites (including helminth eggs and larvae), vaginal clue cells, motile organisms (eg, Trichomonas), and syphilis (via darkfield microscopy). Visibility of fungi can be increased by applying 10% potassium hydroxide (KOH) to dissolve surrounding tissues and nonfungal organisms (Fredricks and Relman, 1999). The clinician orders a stain based on the likely pathogens, but no stain is 100% specific. Most samples are treated with Gram stain and, if mycobacteria are suspected, an acid-fast stain. However, some pathogens are not easily visible using these stains; if these pathogens are suspected, different stains or other identification methods are required. Because microscopic detection usually requires a microbe concentration of about 1 × 105/mL, most body fluid specimens (eg, CSF) are concentrated (eg, by centrifugation) before examination. Types of Commonly Used Stains. Gram stain: The Gram stain classifies bacteria according to whether they retain crystal violet stain (Gram-positive—blue) or not (Gram-negative—red) and highlights cell morphology (eg, bacilli, cocci) and cell arrangement (eg, clumps, chains, diploids). Such characteristics can direct antibiotic therapy pending definitive identification. To do a Gram stain, technicians heat-fix specimen material to a slide and stain it by sequential exposure to Gram's crystal violet, iodine, decolorizer, and counterstain (typically safranin). Acid-fast and moderate (modified) acid-fast stains: These stains are used to identify acid-fast organisms (Mycobacterium sp) and moderately acid-fast organisms (primarily Nocardia sp). These stains are also useful for staining Rhodococcus and related genera, as well as oocysts of some parasites (eg, Cryptosporidium). Although detection of mycobacteria in sputum requires only about 5, 000 to 10, 000 organisms/mL, mycobacteria are often present in lower levels, so sensitivity is limited. Usually, several mL of sputum are decontaminated with Na hydroxide and concentrated by centrifugation for acid-fast staining. Specificity is better, although some moderately acid-fast organisms are difficult to distinguish from mycobacteria. Fluorescent stains: These stains allow detection at lower concentrations (1 × 104 cells/mL). Examples are acridine orange (bacteria and fungi), auramine-rhodamine and auramine O (mycobacteria), and calcofluor white (fungi, especially dermatophytes). Coupling a fluorescent dye to an antibody directed at a pathogen (direct or indirect immunofluorescence) should theoretically increase sensitivity and specificity. However, these tests are difficult to read and interpret, and few (eg, Pneumocystis and Legionella direct fluorescent antibody tests) are commercially available and commonly used. India ink (colloidal carbon) stain: This stain is used to detect mainly Cryptococcus neoformans and other encapsulated fungi in a cell suspension (eg, CSF sediment). The background field, rather than the organism itself, is stained, which makes any capsule around the organism visible as a halo. In CSF, the test is not as sensitive as cryptococcal antigen. Specificity is also limited; leukocytes may appear encapsulated. Wright's stain and Giemsa stain: These stains are used for detection of parasites in blood, Histoplasma capsulatum in phagocytes and tissue cells, intracellular inclusions formed by viruses and chlamydia, trophozoites of Pneumocystis jiroveci, and some intracellular bacteria. Trichrome stain (Gomori-Wheatley stain) and iron hematoxylin stain: These stains are used to detect intestinal protozoa. The Gomori-Wheatley stain is used to detect microsporidia. It may miss helminth eggs and larvae and does not reliably identify Cryptosporidium. Fungi and human cells take up the stain. The iron hematoxylin stain differentially stains cells, cell inclusions, and nuclei. Helminth eggs may stain too dark to permit identification. Disadvantages of Microscopic methods: (a) Microscopy may suggest an etiologic agent, but it rarely provides definitive evidence of infection by a particular species. (b) Microscopic findings regarding bacterial morphology may be misleading, because many species can be pleomorphic and conclusions can be influenced by subjective interpretation of the investigator. (c) Limited sensitivity is because a relatively large number of microbial cells are required before they are seen under microscopy (e.g. 104 bacterial cells/ml of fluid) (Fredricks & Relman, 1999). Some micro-organisms can even require appropriate stains and/or approaches to become visible. (d) Limited specificity is because our inability to speciate micro-organisms based on their morphology and staining patterns. II-Culture Methods Culture is microbial growth on or in a nutritional solid or liquid medium; increased numbers of organisms simplify identification. Culture also facilitates testing of antimicrobial susceptibility (Relman DA., 2002) Communication with the laboratory is essential. Although most specimens are placed on general purpose media (e g, blood or chocolate agar), some pathogens require inclusion of specific nutrients and inhibitors or other special conditions (Wade W., 2002) For more than a century, cultivation using artificial growth media has been the standard diagnostic test in infectious diseases. The microbiota associated with different sites in the human body has been extensively and frequently defined by studies using cultivation approaches. The success in cultivation of important pathogenic bacteria probably led microbiologists to feel satisfied with and optimistic about their results and to recognize that there is no death of known pathogens (Relman, 1992 and Wade W, 2002). But should we be so complacent with what we know about human pathogens? Making micro-organisms grow under laboratory conditions presupposes some knowledge of their growth requirements. Nevertheless, very little is known about the specific growth factors that are utilized by innumerous micro-organisms to survive in virtually all habitats, including within the human body (Wade, 2002). A huge proportion of the microbial species in nature are difficult to be tamed in the laboratory. Certain bacteria are fastidious or even impossible to cultivate. Some well-known human pathogens, such as Mycobacterium leprae and Treponema pallidum continue to defy scientists regarding their cultivation under laboratory conditions (Fredricks and Relman, 1999) need more read Recent advances in diagnosis of infectious diseases http://www.amazon.co.uk/Advances-Diagnosis-Infectious-Diseases-Laboratory/dp/3848445751

Wednesday, May 9, 2012

Lectures in Clinical Microbiology: http://www.ndpublisher.in/JAM_Guide.htm

Lectures in Clinical Microbiology: http://www.ndpublisher.in/JAM_Guide.htm: Journal of advances in medicine is new peer review journal submit your article now and get rapid publication http://www.ndpublisher.in/JAM_...

Lectures in Clinical Microbiology: Biohazard Management in Laboratory

Lectures in Clinical Microbiology: Biohazard Management in Laboratory: Standard laboratories biosafety measures: The guidance and recommendations given as minimum requirements pertaining to laboratories of al...

Lectures in Clinical Microbiology: Meningitis

Lectures in Clinical Microbiology: Meningitis: Meningitis Meningitis and encephalitis are potentially life threatening infections especially in children. Meningitis is...

Meningitis


Meningitis Meningitis and encephalitis are potentially life threatening infections especially in children. Meningitis is defined as an inflammation of the meninges, the tough layer of tissue that surrounds the brain and the spinal cord. Aseptic meningitis (AM) is an inflammation of the meninges which is caused mainly by nonbacterial organisms. AM denotes a clinical syndrome characterized by fever , neck stiffness and may be convulsions with a predominance of lymphocytes in the CSF with negative bacterial culture of the CSF. Viral meningitis occurs as an uncommon complication of systemic viral infection that occurs most frequently in infants and children. Morbidity and mortality depend on the infectious agent, age of the child, general health and prompt diagnosis and treatment. Many etiological agents can cause AM .Viruses are the most frequent causes as Enterovirus,Poliovirus , Coxsackievirus ,Echoviruses,Herpes simplex virus (HSV) ,Varicella-zoster virus( VZV) ,Cytomegalovirus (CMV) , Epstein-Barr virus (EBV). Bacterial infections such as tuberculosis , mycoplasma and leptospira can also cause AM. Non infectious causes may include postvaccination with MMR ,rabies and poliomylities vaccines , drugs as nonsteroidal anti-inflammatory drugs (NSAIDs) and antibiotics as amoxicillin, trimethoprim–sulfamethoxazole. Also fungi as Candida , Cryptococcus and parasites as Toxoplasma gondii , Trichinosis , Neurocysticercosis and Naeglari are infrequent causes of AM. Enteroviruses (EVs) especially non polio EVs (NPEV) including echovirus and coxsackie A and B viruses are the most common etiologic agents of AM . EVs meningitis can mimic bacterial meningitis. So, it is important to distinguish AM, meningoen¬cephalitis from bacterial meningitis which demands prompt therapeutic approach and to avoid unnecessary hospitalization and antibacterial treatment in cases AM. Laboratory diagnosis of AM depends on nonspecific tests as macroscopical examination , cell count both total and differential leucocytic count,direct Gram stained ,cell culture and biochemical tests as glucose , protein ,CSF lactate , C-Reactive protein and CSF adenosine deaminase (ADA). The specific tests are virus isolation, virus antigen detection, virus nucleic acid detection and virus antibody detection (serology). Virus isolation is the current method of choice for the diagnosis but it takes several days to be conclusive. However, attempts to isolate virus from CSF are frequently unsuccessful because of the low viral titer in clinical specimens and several types of viruses do not grow well or not at all in tissue culture . Virus antigen detection is more rapid but still manually intensive and relatively insensitive. Virus serology is an indirect approach with many limitations. It is used to assess immune status and to detect the viruses which cannot be cultivated in cell culture.It includes indirect fluorescent antibody testing and enzyme-linked immunosorbent assays (ELISA) which detect antibodies against viruses. The performance of viral serology is useful in the diagnosis of recent, past or chronic viral infections. However , ELISA is not conclusive in the diagnosis of acute EV or HSV meningitis as IgM antibodies may persist for months and it is not unique to the primary phase. There is a great clinical need to develop rapid and sensitive virus diagnostic techniques. Molecular diagnosis may have a significant benefit . PCR is an in vitro method for specific or target cDNA or RNA amplification. The target DNA-or RNA is derived from clinical specimens or a microbial culture. The major advantages of PCR are its rapidity, sensitivity and robustness. But the major disadvantages of PCR are short size,limiting amounts of product and infidelity of DNA replication . The specificity of PCR is based on the sequence of the two primers. This means that any segment of genomic DNA or RNA is a potential target for PCR diagnostic assay. An ideal target sequence should be found in all strains of the virus of interest but not found in any other viruses. Conclusions : * Meningitis is the most common infectious CNS syndrome which is a life – threatening condition especially in children . * Aseptic meningitis is caused mainly by viruses as Enterovirus , Polioviruses , Coxsackievirus ,Herpes simplex virus ,Varicella-zoster virus,Cytomegalovirus.Other causes may include bacterial as tuberculosis , mycoplasma and leptospira or followed MMR ,rabies and poliomyelitis vaccines.Also , drugs as nonsteroidal anti-inflammatory , trimethoprim or fungi as Candida , Cryptococcus and parasites as Toxoplasma gondii , Trichinosis can cause aseptic meningitis. * Enteroviruses are the most common causes of viral meningitis causing appreciable morbidity. * Molecular biological methods as PCR has been developed. It becomes the technique of choice for detecting viral or other pathogen genome. Laboratory Diagnosis of Meningitis Made Ridiculously easy [Kindle Edition] http://www.amazon.co.uk/Laboratory-Diagnosis-Meningitis-Ridiculously-ebook/dp/B007GIXXR6

Monday, May 7, 2012

Biohazard Management in Laboratory

Standard laboratories biosafety measures: The guidance and recommendations given as minimum requirements pertaining to laboratories of all biosafety levels are directed at micro-organisms in risk levels 1–4. Although some of the precautions may appear to be unnecessary for some organisms in risk group 1, they are desirable for training purposes to promote good (i.e. safe) microbiological techniques (GMT) Standard laboratory design and facilities In designing a laboratory and assigning certain types of work to it, special attention should be paid to conditions that are known to pose safety problems. These include: 1. formation of aerosols, 2. work with large volumes and/or high concentrations of micro-organisms, 3. overcrowding and too much equipment, 4. infestation with rodents and arthropods, 5. unauthorized entrance and 6. workflow: use of specific samples and reagents For proper design, wide space must be provided for the safe conduct of laboratory work for cleaning and maintenance. Walls, ceilings and floors should be smooth, easy to clean, impermeable to liquids and resistant to the chemicals and disinfectants normally used in the laboratory. Floors should be slip-resistant. Bench tops should be impervious to water and resistant to disinfectants, acids, alkalis, organic solvents and moderate heat Laboratory illumination should be adequate for all activities. Undesirable reflections and glare should be avoided. Laboratory furniture should be sturdy. Open spaces between and under benches, cabinets and equipment should be accessible for cleaning. While storage space must be adequate to hold supplies for immediate use and thus prevent clutter on bench tops and in aisles. Additional long-term storage space, conveniently located outside the laboratory working areas, should also be provided. Facilities for eating and drinking and for rest should be provided outside the laboratory working areas. Hand-washing basins, with running water if possible, should be provided in each laboratory room, preferably near the exit door. Safety systems should cover fire, electrical emergencies, emergency shower, eyewash facilities and first-aid areas or rooms suitably equipped and readily accessible should be available ). In the planning of new facilities, consideration should be given to the provision of mechanical ventilation systems that provide an inward flow of air without recirculation. If there is no mechanical ventilation, windows should be able to be opened and should be fitted with arthropod-proof screens. In BSL-2, an autoclave or other means of decontamination should be available in appropriate proximity to the laboratory (). In BSL-3, the laboratory must be separated from the areas that are open to unrestricted traffic flow within the building. Additional separation may be achieved by placing the laboratory at the blind end of a corridor, or constructing a partition and door or access through an anteroom (e.g. a double-door entry), describing a specific area designed to maintain the pressure differential between the laboratory and its adjacent space. The anteroom should have facilities for separating clean and dirty clothing and a shower may also be necessary (). Anteroom doors in BSL-3 may be self-closing and interlocking so that only one door is open at a time. A break-through panel may be provided for emergency exit use. Surfaces of walls, floors and ceilings should be water-resistant and easy to clean. Openings through these surfaces (e.g. for service pipes) should be sealed to facilitate decontamination of the room(s) (In BSL-3, the laboratory room must be sealable for decontamination. Air-ducting systems must be constructed to permit gaseous decontamination. Windows must be closed, sealed and break-resistant. There must be a controlled ventilation system that maintains a directional airflow into the laboratory room. A visual monitoring device with or without alarm(s) should be installed so that staff can at all times ensure that proper directional airflow into the laboratory room is maintained (). The building ventilation system must be so constructed that air from the containment laboratory BSL-3 is not recirculated to other areas within the building. Air may be high-efficiency particulate air (HEPA) filtered, reconditioned and recirculated within that laboratory. When exhaust air from the laboratory (other than from biological safety cabinets) is discharged to the outside of the building, it must be dispersed away from occupied buildings and air intakes. Depending on the agents in use, this air may be discharged through HEPA filters. A heating ventilation and air-conditioning (HVAC) control system may be installed to prevent sustained positive pressurization of the laboratory. Consideration should be given to the installation of audible or clearly visible alarms to notify personnel of HVAC system failure (). In BSL-4, the features of a containment laboratory BSL-3 are also applied to a maximum containment laboratory BSL-4 with the addition to class III cabinet laboratory. Passage through a minimum of two doors prior to entering the rooms containing the Class III biological safety cabinet(s) (cabinet room) is required (). Standard Code of practice: This code is a listing of the most essential laboratory practices and procedures that are basic to GMT. In many laboratories and national laboratory programmes, this code may be used to develop written practices and procedures for safe laboratory operations (). Laboratory personnel protection can be fulfilled by coveralls, gowns or uniforms must be worn at all times for work in the laboratory. Appropriate gloves must be worn for all procedures that may involve direct or accidental contact with blood, body fluids and other potentially infectious materials or infected animals. After use, gloves should be removed and hands must then be washed). For proper safety, personnel must wash their hands after handling infectious materials and before they leave the laboratory working areas. Safety glasses, face shields (visors) or other protective devices must be worn when it is necessary to protect the eyes and face from splashes, impacting objects and sources of artificial ultraviolet radiation (). For GMT, pipetting by mouth must be strictly forbidden. Materials must not be placed in the mouth. All technical procedures should be performed in a way that minimizes the formation of aerosols and droplets. The use of hypodermic needles and syringes should be limited. They must not be used as substitutes for pipetting devices or for any purpose other than parenteral injection or aspiration of fluids from laboratory animals (Also, all spills, accidents and overt or potential exposures to infectious materials must be reported to the laboratory supervisor. A written record of such accidents and incidents should be maintained. A written procedure for the clean-up of all spills must be developed and followed. Contaminated liquids must be decontaminated (chemically or physically) before discharge to the sanitary sewer. An effluent treatment system may be required, depending on the risk assessment for the agent(s) being handled (). The laboratory working areas should be kept neat, clean and free of materials that are not pertinent to the work. Work surfaces must be decontaminated after any spill of potentially dangerous material and at the end of the working day. All contaminated materials, specimens and cultures must be decontaminated before disposal or cleaning for reuse. Packing and transportation must follow applicable national and/or international regulations. When windows can be opened, they should be fitted with arthropod-proof screens ( In BSL-3, the code of practice for basic laboratories BSL-1 and 2 is applied with modifications as follows; laboratory protective clothing must be of the type with solid-front or wrap-around gowns, scrub suits, coveralls, head covering and, where appropriate, shoe covers or dedicated shoes. Laboratory protective clothing must not be worn outside the laboratory and it must be decontaminated before it is laundered. The removal of street clothing and change into dedicated laboratory clothing may be warranted when working with certain agents (e.g. zoonotic agents). Also in BSL-3, open manipulations of all potentially infectious material must be conducted within a biological safety cabinet or other primary containment device. Respiratory protective equipment may be necessary for some laboratory procedures or working with animals infected with certain pathogens. Because of the great complexity of the work in the BSL-4 laboratory, a separate detailed work manual should be developed and tested in training exercises. In addition, an emergency programme must be devised. In the preparation of this programme, active cooperation with national and local health authorities should be established). Essential biosafety equipment: Equipment should be selected to take account of certain general principles, i.e. it should be designed to prevent or limit contact between the operator and the infectious material. Also, it should be constructed of materials that are impermeable to liquids, resistant to corrosion and meet structural requirements and fabricated to be free of burrs, sharp edges and unguarded moving parts. Essential biosafety equipment include pipetting aids to avoid mouth pipetting. Also, biological safety cabinets should be used whenever infectious materials are subjected to centrifugation, grinding, vigorous shaking or mixing, sonic disruption and opening of containers of infectious materials whose internal pressure may be different from the ambient pressure. Plastic disposable transfer loops also should be available. Alternatively, electric transfer loop incinerators may be used inside the biological safety cabinet to reduce aerosol production. Screw-capped tubes and bottles, autoclaves or other appropriate means to decontaminate infectious materials are also required (