Lab Safety

Lab Safety – VHP Safety Basics: Vapor phase hydrogen peroxide decontamination technology is relatively new, having been available since 1991. Vaporized hydrogen peroxide (VHP), or hydrogen peroxide vapor (HPV), is gaining popularity, and there has been a rapid expansion of its use. Laborator source: nice article you can read http://www.pharmamicro.com/2012/10/laboratory-safety-vhp.html?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+Pharmig+%28Pharmig%29&utm_content=FaceBook

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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 –70ºC 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

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

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 (

(I) Septic meningitis Septic meningitis is an inflammation of the meninges caused by bacterial pathogens . Acute meningitis is usually bacterial infection caused by one of several organisms. For development of septic (bacterial) meningitis , the invading organism must gain access to the subarachnoid space (Ashwal ; 1995 ). (A) Pathophysiology : After initial colonization in the upper respiratory tract the invading organism can gain access to the subarachnoid space usually via haematogenous spread and less frequently through an injury such as skull fracture which can cause meningeal seeding via direct bacterial inoculation during trauma (Johansson & Bergentoft ,2005). Bacterial meningitis in the newborn is transmitted either vertically from colonized pathogens in the maternal intestinal or genital tract or horizontally from nursery personnel or other contacts with the patient (Kelley ; 1996).Once bacteria reached to the CSF the relative lack of antibody, complement and white blood cells (WBCs) allow the bacterial infection to flourish (Waler & Rathore; 1995). The types of bacteria that cause septic meningitis vary according to the age of patients. In premature babies and newborn up to three months, the commonest bacteria are group B streptococcus especially in the first week of life–and bacteria that normally inhabit the digestive tract such as Escherichia coli. Listeria monocytogenes may affect the newborn and occurs in epidemics ( Sáez-Llorens & McCracken , 2003). Older children are more commonly affected by Neisseria meningitidis , Streptococcus pneumonia and Haemophilus influenza type B (Ginsberg ,2004). In adults, N. meningitidis and S. pneumonia together cause 80% of all cases of meningitis with increased risk of L. monocytogenes in those over 50 years. Staphylococci , Pseudomonas and other Gram-negative bacilli are likely to cause meningitis especially in patients with trauma, neurosurgical interferences and with impaired immune system (van de Beek et al ., 2006 & Tunkel et al ., 2004 ). ( B) Symptoms and signs : The history in children with bacterial meningitis varies with age. The younger the child, the less likely to exhibit the classical symptoms which include fever, headache and meningeal signs. Babies under 3 months may have very non specific symptoms including hyper or hypothermia, changes in sleeping or eating habits, irritability or lethargy, vomiting, high pitched cry or seizures (Nigrovic et al ., 2007). According to Kelley;(1996) , signs of meningeal irritation are diagnostically helpful when present, they include: * Nuchal rigidity or discomfort on neck flexion. * Kernig's sign which is characterized by passive knee extension in supine patient elicits neck pain. * Brudziniski's sign which is characterized by passive neck , single or both hip flexion . * Meningismus and bulging fontanels may be seen. Other manifestations are systemic findings such as extracranial infection as sinusitis, otitis media, urinary tract infection , arthritis , non-branching pitechae, cutaneous hemorrhage and endotoxic shock especially with Neisseria meningitidis (Thomas et al., 2002) . (C) Risk factors : Risk factors include young age, rapid onset of illness, low peripheral WBCs count and high CSF protein (Pfister , 2005 ). (D) Prognosis : Prognosis depend on the virulence of pathogen , age and severity of acute illness (Puopolo et al ., 2005). In general, mortality rates vary with age and pathogen with the highest being for S. pneumonia. Bacterial meningitis also causes long-term sequelae and results in significant morbidity beyond the neonatal period. Mortality rates are highest during the first year of life, decreasing in mid life and increasing again in elderly persons ( Ray et al ., 2007). (E) complications : *Seizures: Persistent , focal or appear late in the course of disease are more likely to be associated with neurological sequelaes (Chavez-Bueno & McCracken, 2005). *Other complications: include subdural effusions and brain abscesses. Subdural effusions are generally asymptomatic and resolve without neurological sequelae (Prasad , Karlupia , 2007 & Nelson , McCracken , 2005).CNS sequelaes include nerve deafness, cortical blindness, hemiparesis, quadriparesis, muscular hypertonia, ataxia, complex seizure disorders, mental motor retardation, learning disabilities, obstructive hydrocephalus, and cerebral atrophy (Nigrovic et al ., 2007). (II) Aspetic meningitis: According to Lee & Davies ,( 2007) AM is an inflammation of the meninges caused mainly by nonbacterial organisms or other disease processes. AM denotes a clinical syndrome with a predominance of lymphocytes in the CSF in absence of bacterial agents in CSF. Also , AM is an acute infection of the central nervous system that occurs most frequently in infants and young children (Berlin, et al.,1993). It could be either viral, fungal , tuberculous meningitis or other causes ( Rorabough et al ;1993). (A) Pathophysiology: Organisms colonize and penetrate the nasopharyngeal or oropharyngeal mucosa , survive and multiply in the blood stream and they invade host immunological mechanisms and spread through the blood-brain barrier. Infection cannot occur until colonization of the host has taken place (usually in the upper respiratory tract).The mechanisms by which circulating viruses penetrate the blood-brain barrier and seed the CSF to cause meningitis are unclear ( CDC ; 2005). In tuberculous meningitis infection begins in the lung and may spread to the meninges by a variety of routes.Blood-borne spread occurs and 25% of patients with miliary TB have TB meningitis by crossing the blood-brain barrier.A proportionof patients may get TB meningitis from rupture of a cortical focus in the brain (Rich focus) and little proportions get it from rupture of a bony focus in the spine. It is rare and unusual for TB of the spine to cause TB meningitis , but isolated cases have been described(Jain et al .,2006). (B) Symptoms and signs: Clinical presentations and courses of AM are markedly variable. Severity of presentations are correlated with prognosis. They usually start by general viral prodroma for several days which is presented by fever, headache, nausea, vomiting , lethargy, myalgia, dysuria and pyuria (Kung., 2007). Specific prodroma in cases of Varicella zoster virus (VZV), Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Measles and Mumps viruses have been reported such as rash, lymphadenopathy, hepatosplenomegaly and parotid enlargement.Common presentation of viral meningitis and encephalitis is encephalopathy with diffuse neurological symptoms and signs including behavioral and personality changes and/or altered mental status, decreased consciousness, generalized seizures, acute confusional or amnesia states but meningismus and headache are less common. Other nonspecific symptoms may include arthralgia, myalgia, sore throat, weakness and lethargy (Moran et al; 2003). Focal signs such as cranial nerve defects, hemiparesis, focal seizures and autonomic dysfunction are encountered. Other signs of viral meningitis include ataxia , dysphagia and hydrophobia have been reported especially in rabies (Lee et al.2006) . The clinical symptoms of AM are characterized by fever (>38◦C), malaise, vomiting and in some cases petechial rashes. Signs of meningeal irritation include neck stiffness,Kernig’s sign and Brudzinski’s sign .These signs are poorly noted in adults . The nonspecific nature of the symptoms and clinical signs mean that we must confirm the diagnosis (Thomas, 2002) . A variety of clinical manifestations which are associated with enteroviral infections , include respiratory illness, acute hemorrhagic conjunctivitis, myocarditis, neonatal sepsis-like disease, encephalitis and acute flaccid paralysis (Jen-Ren et al;2002) . The clinical features of tuberculous meningitis are non-specific , therefore the discrimination of cases of tuberculous meningitis from other causes of meningitis by clinical features alone is often impossible (Thwaites & Tran, 2005). (C) Epidemiology : The epidemiology of AM and other CNS infections are not fixed and may vary according to location and season (Tyler ,2004). Viral meningitis occurs worldwide as epidemics and as sporadic cases. Cases of enteroviral meningitis are observed increased in the late summer (Tyler ,2004). The incidence of tuberculous meningitis(TBM) is related to the prevalence of TB in the community, and it is still the most common type of chronic CNS infection in developing countries. TB is the seventh leading cause of death and disability worldwide ( Dinnes et al ., 2007). (D) Etiology of aseptic meningitis : Many etiological agents are responsible for AM. Viruses are the most frequent causes. 1-Viruses: a-Enterovirus :Enteroviruses(EVs)belong to the family of Picornaviridae. EVs are small, non enveloped, single-stranded RNA viruses that have been classified into 68 serotypes (Stanway et al.,2005) . EVs include polioviruses and non polioviruses which are including echovirus and coxsackievirus(Andreoletti et al. 1998).All the EVs groups are the common etiological agents of AM (Leite & Barbosa , 2005). Enteroviral meningitis is commonly diagnosed in children less than 5 years of age. It is a febrile illness with anorexia and general malaise or may present as a rather abrupt onset of fever, nausea and headache. It could be followed by meningeal signs with stiffness of the neck or back and muscle weakness may occur which is clinically similar to mild poliomyelitis (Yerly et al; 1996). b-Echovirus : Echovirus is RNA virus that belongs to the genus enterovirus(non polio type) of the picornaviridae family. Echovirus is found in the gastrointestinal tract and hence it is being part of the enterovirus genus (Hauri et al ., 2005). Echovirus is highly infectious and its primary target is children. The echovirus is among the leading causes of acute febrile illness in infants and young children ( Chen et al ., 2005). Echovirus causes a nonspecific exanthemas, herpangina which is a fever and skin rashes that may be maculopapular, morbilliform, macular, petechial or papulopustular in nature ( Pichichero et al ., 1998). Severe forms of disease may be complicated by meningitis especially in infants younger than 3 months and encephalitis, neonatal sepsis and myocarditis especially in patients with altered immunity (Paananen et al ., 2003). c-Herpesvirus : Human herpes viruses include herpes simplex virus (HSV) type I (HSVl ) and type 2 (HSV2),VZV,CMV, Human herpesvirus 6 (HHV6) and EBV(Cardone et al., 2007). HSV AM is most commonly associated with primary genital infection with HSV type 2. Acute AM has also been associated with VZV in patients with or without typical skin lesions. Cases of recurrent Mollaret's meningitis have been associated with HSV type 1, HSV type 2 and EBV( Subramanian & Geraghty, 2007). The structure of herpes virus is spherical in shape. Its lipid envelope encloses the nucleocapsid that arranged in a icosahedral form. Its genome is a single, linear, double-stranded molecule. The capsid is surrounded by a number of loosely associated proteins known collectively as the tegument. Many of these proteins play critical roles in initiating the process of virus reproduction in the infected cell. The tegument is in turn covered by a lipid envelope studded with glycoproteins that are displayed on the exterior of the virion (Mettenleiter et al.,2006). The majority of patients with HSV meningitis present with subacute neurological symptoms developing over 1-7 days and the classic symptoms include: headache, neck stiffness, fever, chills, photophobia with other common meningeal symptoms such as vomiting, seizures and altered consciousness. These manifestations are not evident in infants and elderly ( Fatahzadeh &Schwartz,2007) . Figure 1 :Structure of herpesvirs (Roizman et al ., 2006) d-Cytomegalovirus : Cytomegalovirus (CMV) is a viral genus of the herpesviruses group in humans and it is commonly known as human cytomegalovirus (HCMV) or human herpesvirus 5 (HHV-5) (Adler , 2005). All herpesviruses share a characteristic ability to remain latent within the body over long periods and its structure is similar to HSV structure (Bennekov et al ., 2004). HCMV infection may be a life threatening condition especially in immunocompromised patients. Active infection in healthy children and adults can cause prolonged high fever, chills, severe tiredness, a generally ill feeling, headache and spleenomegaly.Most infected newborns have no symptoms at birth but in some cases symptoms include poor weight gain, swollen glands, rash, liver, lung and blood involvement (Bottieau et al .,2006). Patients with impaired immune systems are more prone to serious, potentially life-threatening illnesses, with fever, pneumonia, CNS complications as meningitis , encephalitis ,liver involvement and anaemia. Illnesses can last for weeks or months and can be fatal. In persons with human immunodeficiency virus (HIV) infection , CMV can infect the retina of the eye (CMV retinitis) and cause blindness (Griffiths & Walter , 2005). e-Epstein-Barr virus : EBV or human herpesvirus 4 infects more than 95% of the world's population. The most common manifestation of primary infection with this virus is acute infectious mononucleosis(glandular fever) which is a self-limited clinical syndrome and frequently affects adolescents and young adults (Nicholas & Joseph ,2008). A mature infectious viral particle consists of nucleoid , capsid and an envelope. The nucleoid contains linear double-stranded viral DNA. It is surrounded by icosahedral capsid which is constructed of capsomers which are tubular protein subunits.The envelope derived either from the outer membrane or the nuclear membrane of the host cell encloses the capsid and nucleoid ( the nucleocapsid) (Gasser et al., 2007). Classical symptoms of acute infectious mononucleosis include sore throat, fever, headache and myalgia with generalized lymphadenopathy and splenomegaly , mononucleosis with relative and absolute lymphocytosis. Infection with EBV in younger children is usually asymptomatic or mild. However, EBV is also a human tumor virus which is the first virus associated with human malignancy as nasopharyngeal carcinoma and Burkitt lymphoma (Chaganti et al., 2008). Nonfatal complications are encountered as various forms of CNS and hematological affection.CNS affections are in the form of meningitis, encephalitis, hemiplegia and transverse myelitis. Hematological affection as EBV can cause autoimmune hemolytic anemia and various cytopenias ( Lockey et al., 2008). f-Varicella zoster virus : Varicella zoster virus (VZV) or human herpesvirus 3 is a member of eight herpes viruses known to infect humans. It commonly causes chicken-pox in children and both shingles and postherpetic neuralgia in adults (Steiner et al., 2007). VZV is closely related to the herpes simplex viruses (HSV) sharing much genome homology (CDC,1996). Chicken-pox (varicella) may rarely causes complications as meningitis ,encephalitis or pneumonia. VZV remains dormant in the nervous system of the infected person (virus latency) in the trigeminal and dorsal root ganglia. In about 10-20% of cases, VZV reactivates later in life producing a disease known as herpes zoster or shingles. Serious complications of shingles include postherpetic neuralgia, zoster multiplex, myelitis, AM , herpes ophthalmicus (Steiner et al., 2007). g-Measles virus :Measles is one of the typical viral diseases of childhood. However, unlike other common viral diseases , measles often leads to severe complications that may be fatal ( Helfand et al. ,2008). Measles virus is a member of the family of Paramyxoviruses which is enveloped,single strand RNA(ssRNA)with helical symmetry.The envelop has only one glycoprotein type which is haemaglutinin (HA-antigen) (Leonard et al., 2008). Figure 2:Structure of measles virus (MicrobiologyBytes , 2008). The classical symptoms of measles include a four day fever up to 40°C ,cough, coryza (runny nose) and conjunctivitis (red eyes). Pathognomonic koplik's spots are seen inside the mouth.The characteristic rashs are classically described as a generalized,maculopapular and erythematous that begin several days after the fever starts. They begin on the head before spreading to cover most of the body and often cause itching. The rashs change colour from red to dark brown before disappearing (Perry et al., 2004). Complications with measles are relatively common ranging from relatively mild to sever include diarrhea , pneumonia , encephalitis , meningitis, corneal ulceration leading to corneal scarring (Torjesen ,2008). h-Mumps virus : Mumps or epidemic parotids is a viral disease of the human species. It is a paramyxovirus with the same genetic structure as Measles virus (Hviid et al ., 2008). It manifested by painful swelling of the salivary glands (classically the parotid gland) . Painful testicular swelling and rash may also occur. The symptoms are generally not severe in children. In teenage males and adults, complications such as infertility or subfertility are more common due to orchitis (testicular inflammation).Central complications include AM which is commonly occurring asymptomatically with inflammatory cells in CSF 50%–60% of patients.Symptomatic meningitis (headache, stiff neck) occurs in up to 15% of patients and resolves without sequelae in 3–10 days (Kanra et al ., 2004). The disease is generally self-limited and the symptoms can be controlled by painkillers druges (Preveden et al ., 1996). i-Rubella Virus : Rubella is commonly known as German measles because the disease was first described by German physicians in the mid-eighteenth century. This disease is often mild and attacks often pass unnoticed. The disease can last one to five days. Children recover more quickly than adults. Infection of the mother by Rubella virus during pregnancy is serious especially in the first 20 weeks of pregnancy as the child may be born with congenital rubella syndrome (CRS) (Richardson et al ., 2001). Rubella virus is the only member of the Rubivirus genus of the Togavirus family. Unlike most Togaviruses it is not arthropod-borne but is acquired via the respiratory route. It is an enveloped (toga=cloak), non-segmented, positive sense, RNA virus (Dayan et al., 2006 & Stegmann, Carey , 2002). After an incubation period of 14-21 days, the primary symptom is the appearance of a rash (exanthema) on the face which spreads to the trunk and limbs and usually fades after three days and the rash disappears after a few days with no staining or peeling of the skin. Other symptoms include low grade fever, post cervical lymphadenopathy, joint pain, headache and conjunctivitis. ( Atreya et al., 2004). Rubella can cause congenital rubella syndrome in the newly born. The syndrome (CRS) follows intrauterine infection by the virus and comprises cardiac, cerebral, ophthalmic and auditory defects. It may also cause prematurity, low birth weight , neonatal thrombocytopenia, anaemia and hepatitis (De Santis et al ., 2006 ). In most cases there is neural involvement , irritability, motor tone problems, mental retardation,meningitis, encephalitis , abnormal posture and neurosensory hearing loss ( Weisinger & Pesudovs ,2002). j- Lymphocytic Choriomeningitis virus : Lymphocytic choriomeningitis or lymphocytic meningoencephalitis (LCM) is a rodent-borne viral infectious disease that may cause AM , encephalitis or meningoencephalitis. The causative agent is the lymphocytic choriomeningitis virus (LCMV) which is a member of the family Arenaviridae which is ssRNA , enveloped virus (Barton & Mets , 2001). In humans LCMV infection usually causes mild illness such as fever, fatigue, loss of appetite, muscle aches, headache, nausea and vomiting. A small number of individuals may become quite ill and develop meningitis or encephalitis .Other individuals may not have any symptoms (Barton & Mets , 2001). Figure 3 : Diagram and electron micrograph of LCMV. International Committee on Taxonomy of Viruses database Management( ICTVdB) , 2006.

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Laboratory Diagnosis of meningitis

Laboratory diagnosis of AM is essential to differentiate between aseptic and septic meningitis. Purulent or septic meningitis is life-threatening but potentially treatable disease whereas viral or AM usually subsides spontaneously (Lee & Davies ,2007).

The identification of specific causes of AM help in prognosis. The EVs are responsible for the majority of cases of AM which need no treatment and have a self limited course, so it is essential to exclude other causes of meningitis such as HSV, VZV, bacterial meningitis to avoid unnecessary hospitalization and the use of antibiotics and therapeutic medications (Rotbart; 1995).

The most important laboratory technique for diagnosis of meningitis is CSF examination which can be considered as a corner stone for differentiation between viral and bacterial meningitis (Lee & Davies ,2007).

Examination of CSF :

(a)-Cerebrospinal fluid:

Cerebrospinal fluid (CSF) : is a clear body fluid that occupies the subarachnoid space and the ventricular system around and inside the brain. Essentially, the brain "floats" in it.More specifically,CSF occupies between the arachnoid mater (the middle layer of the brain cover, meninges) and the pia mater (the layer of the meninges closest to the brain). Moreover , it constitutes the content of all intra-cerebral (inside
the brain, cerebrum) ventricles, cisterns and sulci (singular sulcus) as well as the central canal of the spinal cord. It is an

approximately isotonic solution and acts as a "cushion" or buffer for the cortex. Also providing a basic mechanical and immunological protection to the brain inside the skull )Zakharov et al.,2003).

Amount and constitution:
CSF is produced at a rate of 500 ml/day. Since the brain can only contain from 135-150 ml, large amounts are drained primarily into the blood through arachnoid granulations in the superior sagittal sinus. The CSF contains approximately 0.3% plasma proteins or 15 to 40 mg/dL depending on sampling site. CSF pressure ranges from 60 - 100 mmH2O or 4.4 - 7.3 mmHg with most variations due to coughing or internal compression of jugular veins in the neck ( Dixon et al.,2002).
Function:

CSF has many putative roles including mechanical protection of the brain, distribution of neuroendocrine factors and prevention of brain ischemia. The prevention of brain ischemia is made by decreasing the amount of CSF in the limited space inside the skull. This decreases total intracranial pressure and facilitates blood perfusion (Saunders et al.,1999).

(b) - CSF collection :

CSF is usually collected by an experienced medical officer or health worker by lumbar puncture through aseptically inserting a needle into the subarachinoid space usually at the level of lumber spine .The procedure may be dangerous if the intracranial pressure ( ICP) is raised , so the clinician should be check that there is no papilloedema before the proceeding (Rotbart , 2000 ).

About 6 ml is collected in fresh sterile screw-capped container and divided into 2 parts. A part for culture and should be incubated at 37◦c.The other part should be transported on ice and examined for other tests as cell count, microscopically examination and biochemical analysis including measurement of glucose , protein and for antigen detection (Negrini et al ., 2000).

Lumbar puncture should be avoided in patients with depressed levels of consciousness and shock. Contraindications to lumbar puncture include the following: prolonged or focal seizures , focal neurological signs,widespread purpuric or petechial rash ,pupillary dilatation or asymmetry , impaired oculocephalic reflexes , abnormal posture or movement - decerebrate or decorticate movement or cycling , signs of impending brain herniation ( inappropriate low pulse, raised blood pressure, irregular respiration) , coagulation disorder , papilledema and hypertension (American Academy of Neurology ,2005).

(c) - Storage and transport :

According to Wong et al .,( 2000) the specimens which will be examined for viral detection may be stored by freezing up to 5 days at 4 c and for 6 days or more at -20◦ c after proper mixing in viral transport media. Viral transport media are used to transport small volume of fluid specimen, small tissue, scrapings and swab specimen.These media contain serum , albumin and gelatin to stabilize virus and antimicrobial agents as penicillin,streptomycine and a more potent mixture include vancomycine,gentamycine and amphotricine.CSF is potentially highly infectious and must be handled and transported with great caution .

(I) – Nonspecific discriminatory tests:

A-Macroscopical examination:

Normal CSF is clear and colorless like water. A yellow color "xanthochromic" may result from subarachnoid hemorrhage and traumatic lumbar puncture .



The normal number of WBCs in the CSF in adults vary from 0 to 3 cells/cmm, a maximum of 5 cells/cmm and they are mainly lymphocytes. In children less than one year old , they vary from 0 to 30 cells/cmm and they are mainly polymorphs. Normal CSF contains a small number of lymphocytes and monocytes. The lymphocytes present in the CSF are similar to those in the peripheral blood. Small lymphocytes predominate and 75 to 95% are T lymphocytes (Puccioni-Sohler ,2002 ).


Cell counts over 1000/cmm usually are caused by bacterial infections while counts of 500-1000 /cmm may be bacterial or viral and need further evaluation. In AM the total WBCs count is usually <500/cmm with lymphocytes predominance. However, several studies proved that 20-75% of AM especially those caused by EVs , PMN's were predominant in initial CSF samples (Glimaker et al; 1992). Blood samples should be collected before starting antibiotic therapy for culture and serodiagnosis of viral agents as mumps ,herpes simplex or as Mycoplasma Pneumonia which may be difficult to be cultivated and require serological confirmation of infection (Cinque et al ; 1996) . C-Direct Gram Stained of CSF : Gram staining is a common procedure in the traditional bacteriological laboratory. The technique is used as a tool for the differentiation of Gram-positive and Gram-negative bacteria as a first step to determine the identity of a particular bacterial sample (Beveridge , 1990) . As a general rule Gram-negative bacteria are more pathogenic than Gram-positive bacteria due to their outer membrane structure (Søgaard et al ., 2007). ***Gram-negative bacteria : The proteobacteria are a major group of Gram-negative bacteria. These include many medically relevant Gram-negative cocci, bacilli and many bacteria associated with nosocomial infections (Beveridge ,2001). ***Gram-positive bacteria : They include Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms (Beveridge ,2001). A part of CSF should be centrifuged to get a deposit which stained by Gram stain . The stain may help in diagnosis but the bacterial pathogen may be missed in up to 30% of culture proven cases of bacterial meningitis and is negative in all cases of A.M ( Waler and Rathore ; 1995). According to Saitoh et al. (2005), if a tuberculuse (TB) meningitis is suspected , Zheil-Neelsen smear is done for detection of acid fast bacilli (AFB) and culture on one or two slope of Lowenstein-Jensen medium and incubated for 2-6 weeks at 37◦C .The growing organisms are identified by colonial morphology, staining characters and biochemical reactions . The direct smear examination for acid-fast Mycobacterium is a rapid method but requires a minimum of 104 cells / ml which makes detection extremely difficult for samples such as CSF, pleural effusion and peritoneal fluid ( Kulkarni, et al. 2005). If Yeast cells are seen when performing a cell count or detected in a Gram stain , a small drop of CSF sediment is transferred to a slide with addition of small drop of India ink for the detection of Cryptococcus neoformans if present ( Johansen , et al.2004). D- Culture of CSF: Immediately, part of the CSF deposit seeded heavily on culture media as blood agar, chocolate agar as ( Thayer Martin media ) and incubated in humid air at 37◦C for 24-48 hours plus 5-10% CO2 . Tube of cooked-meat broth and another blood agar plate as Columbia , Brucella or Scheadler should be seeded by deposit and incubated for 2-5 days in an anaerobic atmosphere when there is a suspicious of anaerobic infection. The culture inspected after overnight incubation for identifying any growth , if no growth appears after overnight incubation , the plates are reincubated for another day and reinspected for growth (Nigrovic et al ., 2004 ) . The culture method in tuberculous meningitis is a definitive diagnosis but it is limited by the slow growth rate over several weeks. The sensitivity of the culture method decreases in certain samples with a low number of M. tuberculosis. It could be as low as 40 % for CSF, pleural effusion and peritoneal samples (Schluger et al., 2001). The radiometric method (BACTEC) is based on the measurement of the radioactive CO2 released by metabolism of the radioactive or carbon labeled palmitic acid present in the liquid culture media from mycobacterial growth. This method reduces the culture time, but still requires 7 – 10 days for positive cultures (Bonington et al, 2000). Mycobacteria Growth Indicator Tube (MGIT) system is a non-radiometric method, it has an oxygen sensitive fluorescent sensor embedded in silicone base to serve as an indicator of mycobacterial growth. As the actively growing and respiring mycobacteria consume the dissolved O2, the sensor glows indicating mycobacterial growth. This is observed by using an ultra violet lamp with a wave length of 365 nm (Chitra and Prasad , 2001). Though the use of culture method in leptospirosis confirms diagnosis and also aids in identifying the prevalent serovar, it is rarely used, as it is very tedious, complicated, expensive technically demanding time consuming, requiring prolonged incubation minimum 1 month before declaring a sample negative and may not be successful (low sensitivity). Additionally they are highly infectious organisms requiring strict biosafety facilities (Dutta & Christopher , 2005). Blood or CSF culture for diagnosis of brucellosis in patients with primary infections gives excellent sensitivity results but in individuals with previous contact with the microorganism or occupational exposure and symptoms of acute or persistent infection (very frequent in endemic areas) culture gives poor results( Jordi & Miquel , 2004). The risk of spread of infection, difficulty in culture, time period of more than 6 weeks and positivity of blood or CSF culture in only 50 to 70% patients do not make culture method the investigation of choice for the diagnosis of brucellosis (Kochar et al ., 2000). Diagnosis of Lyme disease by culture method can be done but it is difficult as it requires specialized medium not generally available in most microbiological laboratories and prolonged incubation at relatively low temperatures. Even in specialized labs, the number of spirochetes present in the samples are so low that the yield of culture and even PCR testing are low. The best sensitivity reported in CSF culture in Lyme meningitis is only 10 % as spirochetes are so few in number or even none in the aliquot tested. As a result , diagnosis depends heavily on demonstration of specific anti-B.burgdorferi antibody in serum or CSF (Pachner & Steiner , 2007). Although T. pallidum cannot be grown in culture, there are many tests for the direct and indirect diagnosis of syphilis.Still, there is no single optimal test. Direct diagnostic methods include the detection of T. pallidum by Dark-field microscopy examination of fluid or smears from lesions, histological examination of tissues or nucleic acid amplification methods such as PCR. Indirect diagnosis is based on serological tests for the detection of antibodies. Serological tests are fall into two categories: nontreponemal tests for screening and treponemal tests for confirmation . All nontreponemal tests measure both immunoglobulin (Ig) G and IgM antiphospholipid antibodies formed by the host in response to lipoidal material released by damaged host cells early in infection and to the lipid from the cell surfaces of the treponeme itself (Sam , 2005). In diagnosis of CNS candidiasis ,the significance of a positive culture from the CSF may be unclear. Contamination of the CSF sample may occur because of colonization of the skin or when cultures have been taken from external reservoirs that contain CSF. Several non-culture-based methods have been developed for diagnosing invasive fungal infections of the CNS, such as cryptococcal meningitis and CNS aspergillosis. Similarly, a Candida cell wall component, mannan, has been used as a target for serological tests. Although the detection of circulating mannan was found to be of limited value in the diagnosis of invasive candidiasis, detection of mannan in CSF could be a valuable tool for diagnosing CNS candidiasis (Frans et al ., 2004). E-Biochemical tests : The supernatant part of centrifuged CSF tested for glucose , protein , lactate , C-Reactive protein , adenosine deaminase (ADA) content , estimation of the level of cytokines , lysozyme tests,total and differential leucocytic count ( Salmaso et al ., 1997). (a)- Glucose content : Simultaneous estimation of blood and CSF glucose levels is the most discriminatory test of the nonspecific CSF parameters to differentiate between bacterial and viral meningitis. CSF contain 2.2-4 mmol glucose/liter .Normal CSF glucose is about 60% of serum glucose value. If CSF glucose is <50% of serum glucose this will raise the possibility of bacterial meningitis. Glucose level is usually reduced in bacterial meningitis but may be normal or slight decreased in viral meningitis (Cinque et al ; 1996) . (b)- Protein content : Protein concentration which is below 0.4 g/L in normal CSF is usually more elevated in bacterial meningitis but may be normal or mild elevated in viral meningitis. In purulent (septic) meningitis , the glucose concentration is reduced and the protein concentration increased but in AM , the glucose concentration is normal and the protein concentration either normal or raised a little ( Cinque et al ; 1996). (c)- CSF lactate : The best test to differentiate bacterial from viral meningitis is the measurement of CSF lactate . Lactate levels are particularly important when CSF Gram staining is negative and there is a predominance of PMNs with low glucose in the CSF. CSF lactate concentrations greater than 3.5 mmol/L are characteristic of acute bacterial meningitis. As the lactate concentration in the CSF is independent of that of serum, there is no necessity to test the serum level (Cunha,2004). (d)- Acute phase reactants : Hansson et al.( 1993) found that determination of concentrations of alpha-¬1-acid glycoprotein (AAG) and C-reactive protein (CRP) in serum and alpha-2-ceruloplasmin (CER) in CSF are useful in differentiation between bacterial and viral meningitis. In children younger than 6 years of age , a discriminatory level of serum CRP of 20 mg/L can be used to distinguish between bacterial and viral meningitis but for older patients, a discriminatory level of 50 mg/L is more appropriate. Determination of AAG, CRP in serum are good markers for treatment efficacy and infectious complications in case of bacterial meningitis (Kwaik et al; 1995 and Paradowski et al; 1995) . (e)-Estimation of cytokines : Interleukin 6 (IL6) in CSF was reported to be as a diagnostic marker in the differential diagnosis of meningitis.It can be measured using monoclonal antibody enzyme immunoassay or Radioimmunoassay(RIA). CSF IL6 concentrations were found to be elevated in pyogenic meningitis in 100% of cases and in >50% of viral and other subarachnoid space infections and rarely in patients without CNS infections. Though, patients affected by pyogenic meningitis show the highest levels of CSF IL6 (Lopez-Cortes et al;1997).

(f)- Lysozyme test :

The principle of this test is to add polymexin M sulfate into
the gel bacterial medium. Rapid differential diagnosis of bacterial and viral meningitis with the use of this test is based on different time of the appearance of the lyses areas in bacterial meningitis. The CSF lysozyme activity is detectable within 15-120 min whereas in viral meningitis it manifests 40-50 min later or does not manifest at all. The results are dependant on the time of the CSF collection as more positive results are obtained when CSF samples are early collected (Babich et al ;1992).

Entero-aggregative E.coli (EaggEC).

Entero-aggregative E.coli (EaggEC).
Mathew and Coworkers described afifty reputed group of Enterovirulent E.coli associated with traveler’s diarrhea in Mexico. This new group was associated to more than 40% of HEP-2 cells in tissue culture assays (Janda and Abbott 1998).

Cravioto et al. (1979) observed that EPEC adhere to HEP-2 cells. However, these investigators also showed that many E.coli strains adhere to HEP-2 cells as well and, moreover, that the adherence pattern of EPEC was described as localized adherence (LA), denoting the presence of clusters or microcolonies on the surface of HEP-2 cells. In contrast, non-EPEC did not adhere in the characteristic microcolony morphology, instead displaying a phenotype initially described as (DA) (Nataro et al., 1987a).

The HEP-2 adherence properties of E.coli isolated phenotype can be sub-devided into three categories, (i)Localized (now EPEC), (ii) Aggregative (now EAEC), (iii) Diffuse (now DAEC).

Aggregative adherence (AA) distinguished by prominent, stacked brick autoagglutination of the bacterial cells to each other, which often occurred on the surface of the cells, as well as on the glass coverslip free from the HEP-2 cells.

Diffusely adherent (DA) bacteria were seen dispersed over the surface of the HEP-2 cell, with little aggregation and little adherence to the glass coverslip free from cells (Nataro, 2001).

1.3.12.1. Pathogenesis.
The pathogenicity of EAEC is not thoroughly under stood, however a characteristic histopathological lesion and several candidate virulence factors have been described.; the site of EAEC infection is not known. In an out break of EAEC diarrhea in infants, the illume was shown to be involved (Eslava et al., 1993). The short incubation period of as little as 7 hours, is most consistent with a small bowel site of infection (Nataro et al., 1995a).

Hicks et al. (1996) and Knutton et al. (1992) however shown that EAEC can adhere to small and to an even greater extent to large-bowel section vitro.

EAEC characteristically enhance mucus secretions by the mucosa, with trapping of the bacteria in a bacterium-mucus biofilm (Tzipori et al., 1992), and volenteers fed EAEC develop a diarrhea predominately mucoid in character (Nataro, 2001).

Infection with EAEC has repeatedly been shown to induce cytotoxic effects on the intestinal mucosa. In rabbit and rat ileal loop models (Vial et al., 1988).

EAEC induce a destructive lesion demonstrated by light microscopy. The lesion is characterized by shortening of the villi, hemorrhage necrosis of the villus tips and mild inflammatory response with oedema and mononuclear infiltration of the sub-mucosa (Nataro, 2001).

Thus, diarrheagenic E.coli which adhered to HEP- 2 cells but which were not of EPEC serotypes were termed (enteroadherent E.coli) (Mathewson et al., 1986). The term “enteroadherent” is still frequently used but should now be replaced by the more precise terms enteroaggregative and diffusely adherent (Vial et al., 1988).
EAEC strains are currently defined as E.coli strains that do not secrete entertoxine LT or ST and that adhere to HEP-2 cells in an AA pattern (Nataro et al., 1995a).

1.3.12.2. Cytotoxins.
The toxic effects observed in animal models, human intestinal explains and T84 cells are not accompanied by interalization of the bacteria or by intinate attachment. Therefore, several groups have attempted to identify secreted cytotoxins.

Eslava et al. (1998) have identified 108-Kda cytoxin which elicits destructive lesions in the rat ileal loop. This protein was described by serum from patients infected with EAEC (Baldwin et al., 1992).

1.3.12.3. Epidemiology.
Strains of EAEC belong to very diverse combinations of O and H type, and even within an outbreak of diarrheal disease strains with several different serotypes may be isolated. The diversity of serotypes and pathogenic mechanisms observed suggests that the genes encoding the aggregative phenotype may be accepted readily by pathogenic strains of E.coli causing these bacteria to be considered as EAEC (Green wood et al., 2002).

1.3.12.4. Clinical diagnosis.
The clinical feauters of EAEC are a watery, mucoid secretory diarrheal illness with low-grade fever and little to no vomiting. Beside bloody (Cravioto et al., 1991).

Stiner et al. (1998) have found a large percentage of patients excreting EAEC have detectable fecal lactoferrin and supernormal levels of IL-8 in the stool. This observation suggests that EAEC infection may be accompanied by a suitable form of mucosal inflammation.

1.3.12.5. Detection and diagnosis.
EAEC infection is diagnosed definitively by the isolation of E.coli from the stools of patients and the demonstration of AA pattern in the HEP-2 assay.

1.3.12.5.1. HEP-2 assay.
• HEP-2 cells assay remains the gold standard for detection of EAEC. This test involves allowing strains of E.coli to adhere to cell monolayers in vitro and observing the pattern of adhesion by microscopy. Although tissue culture tests are laborious, the pattern of adhesion remains the key assay for detecting EAEC.
• An aggregative adhesion gene probe has proved useful as a comparatively rapid means of screening strains as a prelude to HEP-2 adhesion test (Nataro and Kaper, 1998 and Green wood et al., 2002).

Entero-aggregative E.coli (EaggEC).

Entero-aggregative E.coli (EaggEC).
Mathew and Coworkers described afifty reputed group of Enterovirulent E.coli associated with traveler’s diarrhea in Mexico. This new group was associated to more than 40% of HEP-2 cells in tissue culture assays (Janda and Abbott 1998).

Cravioto et al. (1979) observed that EPEC adhere to HEP-2 cells. However, these investigators also showed that many E.coli strains adhere to HEP-2 cells as well and, moreover, that the adherence pattern of EPEC was described as localized adherence (LA), denoting the presence of clusters or microcolonies on the surface of HEP-2 cells. In contrast, non-EPEC did not adhere in the characteristic microcolony morphology, instead displaying a phenotype initially described as (DA) (Nataro et al., 1987a).

The HEP-2 adherence properties of E.coli isolated phenotype can be sub-devided into three categories, (i)Localized (now EPEC), (ii) Aggregative (now EAEC), (iii) Diffuse (now DAEC).

Aggregative adherence (AA) distinguished by prominent, stacked brick autoagglutination of the bacterial cells to each other, which often occurred on the surface of the cells, as well as on the glass coverslip free from the HEP-2 cells.

Diffusely adherent (DA) bacteria were seen dispersed over the surface of the HEP-2 cell, with little aggregation and little adherence to the glass coverslip free from cells (Nataro, 2001).

1.3.12.1. Pathogenesis.
The pathogenicity of EAEC is not thoroughly under stood, however a characteristic histopathological lesion and several candidate virulence factors have been described.; the site of EAEC infection is not known. In an out break of EAEC diarrhea in infants, the illume was shown to be involved (Eslava et al., 1993). The short incubation period of as little as 7 hours, is most consistent with a small bowel site of infection (Nataro et al., 1995a).

Hicks et al. (1996) and Knutton et al. (1992) however shown that EAEC can adhere to small and to an even greater extent to large-bowel section vitro.

EAEC characteristically enhance mucus secretions by the mucosa, with trapping of the bacteria in a bacterium-mucus biofilm (Tzipori et al., 1992), and volenteers fed EAEC develop a diarrhea predominately mucoid in character (Nataro, 2001).

Infection with EAEC has repeatedly been shown to induce cytotoxic effects on the intestinal mucosa. In rabbit and rat ileal loop models (Vial et al., 1988).

EAEC induce a destructive lesion demonstrated by light microscopy. The lesion is characterized by shortening of the villi, hemorrhage necrosis of the villus tips and mild inflammatory response with oedema and mononuclear infiltration of the sub-mucosa (Nataro, 2001).

Thus, diarrheagenic E.coli which adhered to HEP- 2 cells but which were not of EPEC serotypes were termed (enteroadherent E.coli) (Mathewson et al., 1986). The term “enteroadherent” is still frequently used but should now be replaced by the more precise terms enteroaggregative and diffusely adherent (Vial et al., 1988).
EAEC strains are currently defined as E.coli strains that do not secrete entertoxine LT or ST and that adhere to HEP-2 cells in an AA pattern (Nataro et al., 1995a).

1.3.12.2. Cytotoxins.
The toxic effects observed in animal models, human intestinal explains and T84 cells are not accompanied by interalization of the bacteria or by intinate attachment. Therefore, several groups have attempted to identify secreted cytotoxins.

Eslava et al. (1998) have identified 108-Kda cytoxin which elicits destructive lesions in the rat ileal loop. This protein was described by serum from patients infected with EAEC (Baldwin et al., 1992).

1.3.12.3. Epidemiology.
Strains of EAEC belong to very diverse combinations of O and H type, and even within an outbreak of diarrheal disease strains with several different serotypes may be isolated. The diversity of serotypes and pathogenic mechanisms observed suggests that the genes encoding the aggregative phenotype may be accepted readily by pathogenic strains of E.coli causing these bacteria to be considered as EAEC (Green wood et al., 2002).

1.3.12.4. Clinical diagnosis.
The clinical feauters of EAEC are a watery, mucoid secretory diarrheal illness with low-grade fever and little to no vomiting. Beside bloody (Cravioto et al., 1991).

Stiner et al. (1998) have found a large percentage of patients excreting EAEC have detectable fecal lactoferrin and supernormal levels of IL-8 in the stool. This observation suggests that EAEC infection may be accompanied by a suitable form of mucosal inflammation.

1.3.12.5. Detection and diagnosis.
EAEC infection is diagnosed definitively by the isolation of E.coli from the stools of patients and the demonstration of AA pattern in the HEP-2 assay.

1.3.12.5.1. HEP-2 assay.
• HEP-2 cells assay remains the gold standard for detection of EAEC. This test involves allowing strains of E.coli to adhere to cell monolayers in vitro and observing the pattern of adhesion by microscopy. Although tissue culture tests are laborious, the pattern of adhesion remains the key assay for detecting EAEC.
• An aggregative adhesion gene probe has proved useful as a comparatively rapid means of screening strains as a prelude to HEP-2 adhesion test (Nataro and Kaper, 1998 and Green wood et al., 2002).

Enterotoxin production

Enterotoxin production.
Shigella and EIEC infections are both characterized by a period of watery diarrhea that precedes the onest of scantly dysenteric stools containing blood and mucus. Indeed, in the majority of infections with EIEC and many with Shigella, only watery diarrhea occurs.

Nataro et al. (1995b) cloned and sequenced a plasmid borne gene from EIEC (designated sen), which encodes a novel protein of predicted size 63k Da. It was shown that a mutation of the sen gene causes a significant diminution of the entertoxic activity of the parent strain. And that the purified sen protein elicits rises in ISC without a significant effect on tissue conductance (Nataro, 2001).

1.3.11.5. Clinical considerations.
The clinical infections is characterized by fever, several abdominal cramps, malaise and watery diarrhea, accompanied by toxemia. Scantly stool containing pus, muces and blood follows the watery diarrhea (Mahon and Manuselis 2000).

1.3.11.6. Detection and diagnosis.
I- EIEC strains may be non motile and do not ferment lactose, cross reactions between Shigella and EIEC O antigens have been seen. Isolates may be mistaken for non pathogenic E.coli although EIEC do not decarboxylate lysine, more than 80% of E.coli decarboxylate lysin, for this reasons cases of diarrheal illness resulting from EIEC may be underreported (Mahon and Manuselis, 2000).

Recently, DNA probes to identify EIEC strains have been studied and compared with the sereny test. DNA probes to screen stool samples for EIEC have developed which eliminate the need for various other tests to identify EIEC.
Also a PCR assay with primers derived from also effective in a multiplex PCR system to identify EIEC strains simultaneously with other E.coli categories (Nataro and Kaper 1998).
II- EIEC strains can be difficult to distinguish from Shigella spp and from other E.coli strains including nonpathogenic strains. In general identification of EIEC entails demonstrating that the organism posses the biochemical profile of E.coli, yet with the genotypic or phenotypic characteristics of Shigella spp. The classical phenotypic assay for Shigella and EIEC identification is the sereny test, which correlate the ability of the strain to invade epithelial cells and spread from cell to cell (Green Wood et al., 2002 and Mahon and Manuselis, 2000).

Entero-invasive E.coli (EIEC).

Entero-invasive E.coli (EIEC).
Enteroinvasive strains of E.coli (EIEC) are strains produce dysentry with direct penetration, invasion, and destruction of the intestinal mucosa. This diarrheal illnesses is very similar to that produced by Shigella. The EIEC infections seem to occur in adults and children alike (Mahon and Manuselis 2000).

EIEC strains are generally lysine decarboxylase negative, non motile, and lactose negative (Brenner et al., 1973). EIEC apparently lack fimbial adhesions but do not possess a specific adhesions that as in Shigella, is thought to be an outer membrane protein. Also, like Shigella, EIEC are invasive organisms. They do not produce LT or ST and, unlike Shigella, they do not produce the Shiga-toxin (Hale et al., 1997).

1.3.11.1. Pathogenesis.
The pathogenesity is resemble to Shigella pathogenesis. Both organisms have shown to invade the colonic epithilium, a phenotype mediated by both plasmid and chromosomal loci. In addition, both EIEC and Shigella Spp elaborate one or more secretory enterotoxins that may play roles in diarrheal pathogenesis (Nataro, 2001).

1.3.11.2. Invasiveness.
The current model of Shigella and EIEC pathogenesis comprises (i) Epithelial cell penetration, (ii) Lysis, of the endocytic vacuole, (iii) intracellualr multiplication, (iv) directional movement through the cytoplasm, and (v) extension into adjacent epithelial cells (Nataro, 2001).

EIEC and Shigella both cause disease by invading intestinal epithelial. Infection is by ingestion, only a small number of bacteria need to be swallowed as they are relatively resistant to gastiric acid and bile and pass readily into the large intestine where they multiply in the gut lumen. The bacteria passes through the overlying mucus layer, attach to the intestinal epithelial cells and are carried into the cell by endocytosis into the endocytic vacuole which then lysis.

The ability to cause the vacuole to lyse is an important virulence attribute, as organisms unable to do this can’t spread to neighbouring cells. After lysis of the vacuole the bacteria multiply within the epithelial cell and kill it. Spread to neighbouring cells leads to tissue destruction and inflammation which cause dysentry. Pathogenicity in Shigellae and EIEC depends on chromosomal and plasmid genes (Green Wood et al., 2002).

1.3.11.3. Epidemiology.
Epidemiology and ecology of EIEC have been poorly studied. Documented EIEC out breaks are usually foodborne or water borne, although person to person transimission does occur. Infections are usually foodborne but there is also evidence of cross infection. The most common serogroup is O124 (Green Wood et al., 2002).

Enteropathogenic E.coli (EPEC).

Enteropathogenic E.coli (EPEC).
The Enteropathogenic E.coli organisms are the oldest known group of coliforms recognized to cause diarrheal disease in humans and has been linked to infant diarrhea in developing world. Once defined solely on the basis of O and H serotypes, EPEC is now defined on the basis of pathogenic charcteristics. Disease is rare in older children and adults, presumably, because of specific O serogroups have been associated with out breaks of EPEC diarrhea (Lewis, 1997).

1.3.10.1. Pathogenesis.
The ability of EPEC strains to cause diarrhea has been confirmed by oral administration of the organisms to babies and incompletely under stool.

Colonilization of the upper part of the small intestine occurs in infantile entiritis associated with EPEC. In many patients, EPEC are seen by electron microscopy to be intimately associated with the mucosal surface and to be partially surrounded by cup like projections (pedestals” of the enterocyte surface and in areas of EPEC attachment the brush border microocilli are lost. Many strains are strongly adhesive to border microvill are lost. Many strains are strongly adhesive to intestinal epithelial cells, and this represents an important pathogenic mechanism. Such strains of E.coli have been termed attaching, effacing E.coli or entero adherent E.coli (EAEC) (Green Wood et al., 2002).

1.3.10.2. Epidemiology.
1.3.10.2.1. Age distribution:
The most notable feature of the epidemiology of disease due to EPEC is the striking age distribution seen in persons infected with this pathogen. EPEC infection is primarily a disease of infants younger than 2 years. As reviewed by Levine and Edelman (Edelman and Levine 1983). Numerous case-control studies in many countries have shown a strong correlation of isolation of EPEC from infants with diarrhea compared to healthy infants. The correlation is strongest with infants younger than 6 months. In children older than 2 years, EPEC can be isolated from healthy and sick individuals, but a statistically significant correlation with disease is usually not found (Nataro and Kaper 1998).

1.3.10.2.2. Transmission and reservoirs:
As with other diarrheagenic E.coli strains, transmission of EPEC is fecal-oral, with contaminated hands, contaminated weaning foods, or contaminated fomites serving as vehicles.

Unless strict decontamination procedures are followed, admission of an infant to a pediatric ward can result in contamination of crib lien, toys, hand towels, scales.

The reservoir of EPEC infection is thought to be symptomatic or asymptomatic children and asymptomatic adults carriers, including mothers and persons who handle infants. Numerous studies have documented the spread of infection through hospitals, nurseries (Bower et al., 1989).

1.3.10.2.3. EPCE in developing countries:
EPEC is a major cause of infants diarrhea in developing countries. Numerous studies have been founds EPEC to be more frequently isolated from infants with diarrhea than from matched healthy controls (Donnenberg 1995). Particulary in the 0 to 6 months age group, EPEC strains are often the most frequently isolated bacterial diarrheal pathogens (Cravioto et al., 1988).

Several studies have shown that breast feeding is protective against diarrhea due to EPEC. Both human colostrum and milk strongly inhibit the adhesion of EPEC to HEP 2 cells in vitro (Camara et al., 1994).

1.3.10.4. Clinical considerations.
EPEC cause primarily acute diarrhea and it was associated with many common symptoms such as watery diarrhea, vomiting and low-grade fever are common symptoms beside malaise (Cohen and Gianella, 1991).

Fecal leukocytes are seen only occasionally, but more sensetive tests for inflammatory diarrhea such as an anti-lactoferrin latex bead agglutination test are frequently positive with EPEC infection.

As with other diarrheal pathogens, the primary goal of treatment of EPEC diarrhea is to prevent dehydration by correcting fluid and electrolyte imblances. Oral rehydration may be sufficient for milder cases, but more severe cases require parenteral rehydration. Correction nutritional imbalance with lactose free formula or breast milk may be insufficient for some severely ill patients, and total parenteral nutrition may be required. A variety of antibiotics have been used to treat EPEC and have proved useful in many cases. There are no vaccines currently available or in clinical trails to prevent disease due to EPEC (Camara et al., 1994).

1.3.10.5. Detection and diagnosis.
The definition of EPEC has changed in recent years as our knowledge of this pathogen has grown. For many years these organisms were defined only by O serogroups, which were subsequently defined to O:H serotypes. This definition changed as additional serotypes were associated with infantile diarrhea (Edelman and Levine 1983).

Citing recent pathogensis data, the second international symposium on EPEC in 1995 reached a consensus on the basic characteristics of EPEC, the most important of these were the A/E histopathology and the absence of Shiga-toxin. Many EHEC strains also produce the A/E lesion, therefore, determining the presence (indicative of EHEC) or absence (indicative of EPEC) of Stx is essential. The sole defining micorbiological characteristic of EPEC, is no longer deemed an essential characteristic of EPEC, although the majority of EPEC strains fall into certain well-recognized O:H serotypes (Green Wood et al., 2002).

There is some debate whether EPEC strains the lack the EAF plasmid are true pathogen or not so it was found that, only EAF-positive strains and not EAF-negative strains were significantly associated with diarrhea (Echeverria et al., 1991).

Furthermore, EAF-negative strains isolated during the course of an epidemiological study could also be derivatives of EHEC that have lost the phasges that encode Stx.

So EPEC: A/E, Stx-negative strains possessing the EAF plasmid would be called “typical EPEC”, while strains doesn’t possess the EAF plasmid would be called “atypical EPEC” (Nataro and Kaper 1998).



1.3.10.6. Diagnostic tests.
Given that EPEC strains, as with other diarrheagenic E.coli strains, are defined on the basis of virulence properties, there are two approaches to the detection of EPEC in the laboratory (i) phenotypic (ii) genotypic. The phenotypic approach requires the use of cell cultures and fluorescence microscopy, and the genotypic method requires the use of DNA hyberdization or PCR (Nataro and Kaper 1998).

Enterotoxigenic E.coli (ETEC).

ETEC is defined as the E.coli strains that elaborate at least one member of two defined groups of enterotoxins: a heat liable toxin (HLT) and aheat stable toxins(HST). The first is a heat-labile enterotoxin (LT) that shows approximately 80% protein sequence identify with the heat-labile cytotoxinic cholera toxin of vibrio cholerae. The second toxin produced by ETEC is a heat-stable with a molecular mass of about 2Kda. Previous studies suggested that ETEC strains carrying both toxins (HLT+ and HST+) predominate clinically followed in decreasing frequency by strains carrying HSt+ only and those carrying HLT+ only. However, a 1996 review of 700ETEC strains collected over a period of more than 30 years found that the relative prevalence of these toxine type I is not different and that individual toxin-producing patterns may be related to serogroup specificity (Sears and Kaper, 1996 and
Tamura et al., 1996).

1.3.9.1. Epidemiology.
ETEC strains are associated with two major clinical syndromes: weaning diarrhea among children in the developing world, and traveler’s diarrhea. The epidemiologic pattern of ETEC disease is determined in large part by a number of factors: (I) Mucosal immunity to ETEC infection develops in exposed individuals, (ii) Even immune asymptomatic individuals may shed large numbers of virulent ETEC organisms in the stool, and (iii) The infection requires a relatively high infectious dose (Nataro and Kaper 1998). These three feautures create a situation in which ETEC contamination of the environment in areas of endemic infections is extremely prevalent, and most infants in such areas will encounter in warm-climate countries are not well under stool, but it seems likely that water contaminated by human (or) animal sewage plays an important part in the spread of infection (Green Wood, 2002).

1.3.9.2. Clinical symptoms.
The most common symptoms associated with ETEC infection are diarrehea (91-97%) and abdominal cramps (80-94%). Stools are typically watery in consistency, often yellow-tinged, and without the presence of mucus, pus or fecal leukocytes (Cohen and Gianella, 1991).

The gastroentritis induced by ETEC infection is typically indicating guishable from secretory diarrheas elicited by other gram-negative enteropathogens. Less often nausea (39-70%), headaches (35-57%), myalgia (50%), weakness (35%). Chills (31%), and low grade fevers (13-22%) are observed (Role et al., 1995).

Vomiting (2-1%) is not commonly associated with ETEC infection, a fact that helps distinguish this illness from gastrointestinal disturbances caused by norwalk virus.

ETEC infections commonly occur in four settings. In less developed countries, ETEC infection predominate in children younger than a years of age. Unlike the relatively mild infections, ETEC gastroentritis in young children in underdeveloped nations can be serve at times, with dehydration and adverse nutritional consequences leading to retardation of normal growth development (Cohen and Gianella, 1991).

Dehydration has also been noted as a consequence of ETEC infection in industrialized countries. As children mature, the incidence of ETEC disease apparently declines, suggesting that immunity may develop in local inhabitants with advancing age (Black et al., 1981).

1.3.9.3. Detection and Diagnosis.
Detection of ETEC has long relied on detection of the enterotoxins (HLT or HST). HST was initially detected in a rabbit ligaled ileal loop assay, but the expense and lack of standarization caused this test to be replaced by the sucking-mouse assay which become the standard test for the presence of HST for many years (Cryan 1990).

The traditional bioassay for detection of HLT involves the use of cell culture, either the Y, adrenal cell assay (or) the chinese hamster ovary (CHO) cell assay (Donta et al., 1974).

DNA probes were found to be useful in the detection of HLT and HST encoding genes in stool and environmental samples. Since that time several advances in ETEC detection have been made, but genetic techniques continue to attract the most detection and use. It should be stressed that there is no perfect test for ETEC decection of colonization factors because of their great number and heterogeneity (Abdul et al., 1994).

Several PCR assays for ETEC are quite sensitive and specific when used directly on clinical samples or on isolated bacterial colonies. A useful adaptation of PCR is the “multiplex” PCR assay in which several PCR primers are combined with the aim of detecting one of several different diarrheagenic E.coli pathotypes in a single reaction (duToit et al., 1993).