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Wednesday, December 21, 2011

Mechanisms of antibiotic action

Antibiotics target structures and pathways that are unique and important to bacteria such as cell wall synthesis, cytoplasmic membrane synthesis, protein synthesis, nucleic acid (DNA or RNA) synthesis and intermediary metabolism (McCallum, 2010).
A-Inhibition of cell wall synthesis:
Bacterial cell wall function and structure:
A cell wall maintains cellular integrity by countering the effects of osmosis when the cell is in a hypotonic solution. If the wall is disrupted, it no longer prevents the cell from bursting as water moves into the cell by osmosis (Dmitriev et al., 2005).
The major structural component of a bacterial cell wall is its peptidoglycan layer. Peptidoglycan is a huge macromolecule composed of polysaccharide chains of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) molecules that are cross linked by short peptide chains extending between NAM subunits. To enlarge or divide, a cell must synthesize more peptidoglycan by adding new NAG and NAM subunits to existing NAG-NAM chains, and the new NAM subunits must then be bonded to neighboring NAM subunits (Meroueh et al., 2006).
You need to read more
Manual of Antibiotics: Method of Actions, Mechanisms of Resistance and Relations to Health Care associated Infections
http://www.amazon.com/Manual-Antibiotics-Mechanisms-Resistance-ebook/dp/B0050VQWXI

Monday, December 19, 2011

Nanobacteria

term that is used in microbiology is nanbacteria. Nanobacteria have been claimed in last few years to cause a wide variety of diseases.
Nanobacteria are mineral-forming, sterile-filterable, slow-growing Gram-negative infectious agents [5]. They are detected in bovine/human blood and urine. Nanobacteria-like particles have been detected in synovial fluids of arthritis patients and were shown to gradually increase in number and in size in culture [6].
According to their 16S rDNA structure, nanobacteria belong to the alpha-2 Proteobacteria, subgroup, which includes the Brucella and Bartonella species. Nanobacterium sanguineum (nanobacteria) is the smallest self-replicating organism ever detected—at 50–500 billionths of a meter, 1/1,000th the size of the smallest previously known bacteria [7].
Nanobacteria have been claimed to be associated with a variety of human diseases manifested with pathological calcification. Their most remarkable characteristic is the formation of carbonate apatite crystals of neutral pH and at physiologic phosphate and calcium concentrations. The extracellular mineralization forms a hard protective shelter for these hardy microorganisms and enables them to survive conditions of physical stress that would be lethal to most other bacterial species. Nanobacteria are associated with human kidney stones and psammona bodies in ovarian cancer. Many researchers have thon the potential role these particles may play in the development of urologic pathology, including polycystic kidney disease, renal calculi, and chronic prostatitis. Recent clinical research targeting these agents has proven effective in treating some patients with refractory category III prostatitis [8].
Kidney stones can be debilitating and recur in 50% of patients within 5 years. Kidney stone formation is considered to be a multifactorial disease in which the defense mechanisms and risk factors are imbalanced in favor of stone formation [9].
One theory is that if nanoparticles accumulate in the kidney, they can form the focus of subsequent growth into larger stones over months to years. Other factors, such as physical chemistry and protein inhibitors of crystal growth, also play a role.
Mineral forming nanobacteria are active nidi that attach to, invade, and damage the urinary epithelium of collecting ducts and papilla forming the calcium phosphate center(s) found in most kidney stones. Scientists at NASA have used multiple techniques to determine that nanobacteria infection multiplies faster in space flight simulated conditions than on earth [9].
Nanobacteria are considered to initiate kidney stone formation as they grow faster in a microgravity environment and may explain why astronauts get kidney stones on space missions. This discovery may prove to be critical for future exploratory missions to the moon and Mars. For further proof to this hypothesis, screening of the nanobacterial antigen and antibody level in flight crew before and after flight would be necessary. This concept also opens the door for new diagnostic and therapeutic techniques addressing nanobacterial infection in kidney stones.
Nanoparticles, isolated from renal stones obtained at the time of surgical resection, have been analyzed and propagated in standard cell culture medium [10]. Nanoparticles were isolated from the majority of renal stones. Isolates were sensitive toward selected metabolic inhibitors and antibiotics and contained conserved bacterial proteins and DNA. These findings suggest that renal stone formation is unlikely to be driven solely by physical chemistry; rather, it is critically influenced by specific proteins and cellular responses, and understanding these events will through lights toward new therapeutic targets. Using high-spatial and energy resolution near-edge x-ray absorption fine structure at the 25-nm spatial scale, it is possible to define a biochemical signature for cultured calcified bacteria, including proteins, polysaccharides, nucleic acids, and hydroxyapatite [11].
These preliminary reports suggest that nanoparticles isolated from human samples share spectroscopic characteristics with calcified proteins.
Nanobacteria is claimed to be associated with cardiovascular diserases. Scientists at the Mayo Clinic have examined surgical specimens from patients with cardiovascular pathology to predict the presence of nanobacteria [12]. Analysis of areas with positive immuno staining identified spheres ranging in size from 30 to 100 nm with a spectral pattern of calcium and phosphorus (high-energy dispersive spectroscopy).
Nano-sized particles cultured from calcified but not from non calcified aneurysms were recognized by a DNA-specific dye, incorporated radiolabeled uridine, and after decalcification, appeared via electron microscopy to contain cell walls. Therefore, nanometer-scale particles can be visualized in and cultured from human calcified cardiovascular tissue. In a further study nanoparticles were found near plaque-filled arteries in animal models. The study suggests that nanoparticles potentially represent a previously unrecognized factor in the development of arteriosclerosis and calcific arterial disease [12].

Wednesday, December 14, 2011

Nanotechnology and Advances in Medicine

Nanotechnology is an emerging technology with enormous potential in information and communication technology, biology and biotechnology, medicine and medical technology. Nanotechnology refers to the a new area of science in which systems are designed and manufactured at the scale of the atom, or the nanometer scale. More specifically nanotechnology deals with structures of less than 100 nanometer (nm). One nm is 1 billionth of a meter. there are two approaches in nanotechnology: bottom-up and top-down. The first approach, the bottom-up, involves manipulating small numbers individual atoms or more complex molecules, into structures typically using minute probes. The second, top-down, approach implies controlling processes to force atoms and molecules to build-up themselves to desired locations and/or structures.
Novel nano- and bio-materials, and nano devices are fabricated and controlled by nanotechnology tools and techniques, which investigate and tune the properties, responses and functions of living and non-living matter, at sizes below 100 nm. The potential medical applications are predominantly in detection, diagnostics (disease diagnosis and imaging), monitoring, and therapeutics. The availability of more durable and better prosthetics and new drug-delivery systems are of great scientific interest and give hope for cancer treatment and minimum invasive treatments for heart disease, diabetes and other diseases. Many novel nanoparticles and nanodevices are expected to be used, with an enormous positive impact on human health. The vision is to improve health by enhancing the efficacy and safety of nanosystems and nanodevices.

Sunday, December 11, 2011

Obesity linked to bacteria

Can bacterial flora in gut be linked to obesity? How?Metabolic syndrome is a group of obesity-related metabolic abnormalities that increase an individual’s risk of developing type 2 diabetes and cardiovascular disease. Here, we show that mice genetically deficient in Toll-like receptor 5 (TLR5), a component of the innate immune system that is expressed in the gut mucosa and that helps defend against infection, exhibit hyperphagia and develop hallmark features of metabolic syndrome, including hyperlipidemia, hypertension, insulin resistance, and increased adiposity. These metabolic changes correlated with changes in the composition of the gut microbiota, and transfer of the gut microbiota from TLR5-deficient mice to wild-type germ-free mice conferred many features of metabolic syndrome to the recipients. Food restriction prevented obesity, but not insulin resistance, in the TLR5-deficient mice. These results support the emerging view that the gut microbiota contributes to metabolic disease and suggest that malfunction of the innate immune system may promote the development of metabolic syndrome.
Coated from Published Online March 4 2010
Science 9 April 2010:
Vol. 328 no. 5975 pp. 228-231
DOI: 10.1126/science.1179721
•Report
Metabolic Syndrome and Altered Gut Microbiota in Mice Lacking Toll-Like Receptor 5
Matam Vijay-Kumar1, Jesse D. Aitken1, Frederic A. Carvalho1, Tyler C. Cullender2, Simon Mwangi3, Shanthi Srinivasan3, Shanthi V. Sitaraman3, Rob Knight4, Ruth E. Ley2 and Andrew T. Gewirtz1,*
+

http://www.sciencemag.org/content/328/5975/228.abstract

Tuesday, December 6, 2011

Guide for Handling Cytotoxic Drugs-What Health care Should Know?

􀀮􀁐􀁅􀁖􀁍􀁆􀀁􀀒􀀁􀂰􀀁􀀪􀁏􀁕􀁓􀁐􀁅􀁖􀁄􀁕􀁊􀁐􀁏􀀁􀁕􀁐􀀁􀁄􀁚􀁕􀁐􀁕􀁐􀁙􀁊􀁄􀀁􀁄􀁐􀁏􀁄􀁆􀁑􀁕􀁔
To ensure all workers are aware of, and understand the risks associated with the handling and
use of cytotoxic drugs and related waste.
Teaching points:
1.1 Risks associated with occupational exposure to cytotoxic drugs and related waste:
• health risks and toxic effects
• reproductive health risks.
1.2 Rationale for use of cytotoxic drug therapy.
1.3 Legislative requirements for the management of cytotoxic hazards, MSDSs, risk assessment, employer
and worker obligations.
1.4 Institutional policies and procedures.
1.5 Definitions; cell replication; drug classifications, pharmacological actions; rationale for use.
Identification of those drugs which are mutagenic, teratogenic and carcinogenic. Cytotoxic drugs as a
class of drugs:
• define ‘carcinogenic’ ‘mutagenic’ and ‘teratogenic’
• concepts of cell replication
• drug classifications and pharmacological action on cellular reproduction.
1.6 Health surveillance for workers working with cytotoxic drugs;
• health assessment of workers after unprotected exposure to cytotoxic drugs:
° rationale
° health assessment required in response to an unprotected exposure
• principles for initial and ongoing health assessment:
° rationale for personnel management
° the purpose of health assessment
° limitations of current health surveillance methods.
1.7 The importance of accurate record keeping (e.g. an activity log, records of spills and penetrating
injuries). Storage requirements for health surveillance documentation to ensure confidentiality,
perpetual safe keeping and retrieval.
1.8 Incidents and spill management
1.9 Safe disposal methods for cytotoxic drugs and related waste. Safe storage, packaging, consigning and
transport of cytotoxic waste:
• the rationale for the identification, segregation and safe handling of cytotoxic waste
• institution policies and procedures as they apply to:
° segregation of cytotoxic waste
° containment of cytotoxic waste
° transport of cytotoxic waste
° management of cytotoxic drug and related waste spills.
1.10 PPE requirements, including, selection, use, fit, maintenance, storage, cleaning and disposal.
􀀮􀁐􀁅􀁖􀁍􀁆􀀁􀀓􀀁􀂰􀀁􀀱􀁓􀁆􀁑􀁂􀁓􀁂􀁕􀁊􀁐􀁏􀀁􀁐􀁇􀀁􀁄􀁚􀁕􀁐􀁕􀁐􀁙􀁊􀁄􀀁􀁅􀁓􀁖􀁈􀁔
To train workers in the safe preparation of cytotoxic drugs.
Teaching points – to include Module 1, plus:
2.1 Facility requirements:
• minimum requirements for a cytotoxic preparation facility as defined by AS 2567-2002:
Laminar flow cytotoxic drug safety cabinets and AS 2639-1994:Laminar flow cytotoxic drug
safety cabinets - Installation and use
106 􀀨􀁖􀁊􀁅􀁆􀀁􀁇􀁐􀁓􀀁􀁉􀁂􀁏􀁅􀁍􀁊􀁏􀁈􀀁􀁄􀁚􀁕􀁐􀁕􀁐􀁙􀁊􀁄􀀁􀁅􀁓􀁖􀁈􀁔􀀁􀁂􀁏􀁅􀀁􀁓􀁆􀁍􀁂􀁕􀁆􀁅􀀁􀁘􀁂􀁔􀁕􀁆􀀁􀀁􀁝􀀁􀀁􀀁􀀸􀁐􀁓􀁌􀁑􀁍􀁂􀁄􀁆􀀁􀀩􀁆􀁂􀁍􀁕􀁉􀀁􀁂􀁏􀁅􀀁􀀴􀁂􀁇􀁆􀁕􀁚􀀁􀀲􀁖􀁆􀁆􀁏􀁔􀁍􀁂􀁏􀁅
• principles of clean spaces, their creation and maintenance as described in AS 1386.1-1989:
Cleanrooms and clean workstations - Principles of clean space control:
° essential elements necessary for the preparation of cytotoxic drugs—clean room, air handling
system, peripheral rooms, cytotoxic dispensing facility (e.g. laminar air flow equipment,
isolators, cytotoxic drugs safety cabinet)
° their function and use
• management of the cytotoxic preparation facility:
° operation of the cytotoxic drugs safety cabinet or isolator
° maintenance of the preparation facility
° approved devices/equipment used in preparing cytotoxic drugs
° management of cytotoxic spills
° management of contaminated waste generated in the preparation of cytotoxic drugs
° preparation records
• certification reports
• activity logs
• pressure differential records
• environmental monitoring.
2.2 Aseptic preparation of a cytotoxic product.
• principles of aseptic preparation of parenteral solutions
• specific requirements for aseptic preparation in cytotoxic drug safety cabinets or isolators.
2.3 Quality assurance measures required for preparation cytotoxic drugs.
2.4 Safe techniques for cytotoxic drugs. Health and safety hazards posed by handling cytotoxic drugs in
powder and liquid form:
• routes of absorption associated with occupational exposure
• hazards involved when cytotoxic drug aerosols are liberated into a workplace.
2.5 Packaging requirements for the safe presentation and receipt of prepared cytotoxic drugs in individual
packing:
• labelling and packaging requirements for the presentation of prepared cytotoxic drug doses
• procedure for dealing with broken tablets and capsules
• rationale for the use of primary (e.g. a syringe closure), secondary (e.g. the spill-proof overwrap
containing the syringe) and tertiary (e.g. the spill-proof outer transport container) containers for
the maintenance of product integrity and its safe handling.
2.6 Safe storage and transport of cytotoxic drugs in concentrated from:
• legislative requirements for the storage and transport of cytotoxic drugs
• rationale for specific procedures essential for the storage and transport of cytotoxic drugs
• risks associated with the different presentations of cytotoxic drugs
• institutional policy and procedures as they apply to receipt of goods, storage of goods, transport
of goods, management of cytotoxic drug and related waste spills.
2.7 PPE requirements:
• function and use of PPE—demonstrate appropriate use of PPE:
° selection
° putting on
° concurrent use
• cleaning, laundry and disposal of used PPE.
2.8 Waste management principles of waste containment and segregation:
• contaminated waste disposal
• contaminated patient waste
• cytotoxic waste storage and transport requirements.
2.9 Management in the community.
2.10 Incidents and spill management.
2.11 Record keeping.
􀀮􀁐􀁅􀁖􀁍􀁆􀀁􀀔􀀁􀂰􀀁􀀢􀁅􀁎􀁊􀁏􀁊􀁔􀁕􀁓􀁂􀁕􀁊􀁐􀁏􀀁􀁐􀁇􀀁􀁄􀁚􀁕􀁐􀁕􀁐􀁙􀁊􀁄􀀁􀁅􀁓􀁖􀁈􀁔
To train workers in the safe administration of cytotoxic drugs.
Teaching points - Module 1, plus
3.1 Risks associated with administration for operator and patient:
􀀨􀁖􀁊􀁅􀁆􀀁􀁇􀁐􀁓􀀁􀁉􀁂􀁏􀁅􀁍􀁊􀁏􀁈􀀁􀁄􀁚􀁕􀁐􀁕􀁐􀁙􀁊􀁄􀀁􀁅􀁓􀁖􀁈􀁔􀀁􀁂􀁏􀁅􀀁􀁓􀁆􀁍􀁂􀁕􀁆􀁅􀀁􀁘􀁂􀁔􀁕􀁆􀀁􀀁􀁝􀀁􀀁􀀁􀀸􀁐􀁓􀁌􀁑􀁍􀁂􀁄􀁆􀀁􀀩􀁆􀁂􀁍􀁕􀁉􀀁􀁂􀁏􀁅􀀁􀀴􀁂􀁇􀁆􀁕􀁚􀀁􀀲􀁖􀁆􀁆􀁏􀁔􀁍􀁂􀁏􀁅 107
• physical and chemical characteristics of these drugs as they pertain to occupational safety:
° differences in potential risk between lyophilized drugs, powdered and liquid filled preparations
° substances requiring protective containment
• cytotoxic drugs and their rationale for use:
° cure, control, prophylaxis and palliation
° drug dosages, routes of administration, delivery methods, calculation of body surface area
° cytotoxic drug protocols.
3.2 Principles of safe handling for all routes of administration. Safe administration techniques for
cytotoxic drugs:
• health and safety hazards posed by handling cytotoxic drugs in liquid form:
° routes of absorption associated with occupational exposure
° hazards involved when cytotoxic drug aerosols are liberated into a workplace
• packaging requirements for the safe presentation and receipt of prepared cytotoxic drugs in
individual packing:
° labelling and packaging requirements for the presentation of prepared cytotoxic drug doses
° procedure for dealing with broken tablets and capsules
° rationale for the use of primary (e.g. a syringe closure), secondary (e.g. the spill-proof
overwrap containing the syringe) and tertiary (e.g. the spill-proof outer transport container)
° containers for the maintenance of product integrity and its safe handling
• identifying safe routes of administration—principles of safe handling and administration of
cytotoxic drug injections:
° demonstrate correct and safe use of cytotoxic drug injectables
° identify variety of routes by which cytotoxic drugs are administered
° identify appropriate blood values and assessments prior to drug administration
• principles of safe handling and administration of cytotoxic drug infusions:
° identify appropriate equipment for management of infusions of cytotoxic drugs
° demonstrate correct technique for the connection and disconnection of cytotoxic drug
administration equipment
• principles of safe handling and administration of oral and topical cytotoxic drugs:
° demonstrate no-touch technique in the administration of oral cytotoxic drug doses
° no-touch application technique and drug containment procedures when applying topical
cytotoxic drugs
• reasons for the selection of differing vascular access techniques for parenteral cytotoxic drugs:
° demonstrate techniques of vein access appropriate for intravenous administration of various
cytotoxic agents
• selection of a vascular access site for vesicant and irritant cytotoxic drug administration:
° identify appropriate vascular access site for the cytotoxic drug used
° identify drugs that are vesicants and those that are irritants
• principles of safe handling and administration of intrathecal cytotoxic drugs:
° identify cytotoxic drugs that can be safely administered by intrathecal route
° calculate intrathecal drug doses
° identify risks associated with accidental intrathecal administration of vinca alkaloids
° identify strategies to reduce risk of accidental intrathecal administration of vinca alkaloids,
including transport, packaging, labelling, checking and administration
• safe procedures for the emergency cessation of cytotoxic drug administration (e.g. adverse
reaction), demonstrate systematic approach to the containment of cytotoxic drugs during
emergency cessation
• management of extravasation—identify critical steps for the management of extravasation
• packaging requirements when transporting cytotoxic drugs within the treatment unit:
° principles of package containment for cytotoxic drug transport within the treatment unit
° transport requirements following the addition of needles to prepared syringes.
3.3 PPE requirements—function and use of PPE—demonstrate appropriate use of PPE:
• selection
• putting on
• concurrent use
• cleaning, laundry and disposal of used PPE.
3.4 Safe disposal methods for cytotoxic agents and equipment involved in administration:
principles of waste containment and segregation as applied to cytotoxic drugs
• appropriate containers required for cytotoxic waste disposal
• understand the principles of waste containment and procedures for the disposal of cytotoxic
sharps
• procedure for disposal of related cytotoxic drug administration equipment
• safe disposal procedure of PPE.
3.5 Incidents and spill management:
• management of cytotoxic drug spills:
° warning and notification requirements for cytotoxic drug spill management:
- isolation and warning procedures
- remedial action in the event of a spill
- procedure for requesting assistance
° PPE requirements for cytotoxic drug spill management
° principles and procedures for cytotoxic drug spill management:
- equipment necessary to contain the cytotoxic spill
- decontamination solutions or substances for cytotoxic containment
- effective use of cytotoxic spill equipment and decontaminants
- containment and disposal of cytotoxic drug spill materials
° action required when an unprotected exposure to workers occurs (e.g. topical, mucous
membrane, or penetrating injury exposure), identify the appropriate health assessment required
in response to unprotected exposure
° post-spill procedures:
- reporting procedures
- health assessment and follow-up.
3.6 Patient education requirements and ethical considerations.
3.7 Patient handling:
• management of contaminated body substances from patients undergoing and following cytotoxic
drug therapy:
° major pathways of body excretion of unchanged cytotoxic drugs or active drug metabolites
° protective period for safe handing of cytotoxic drug body substances:
- standard excretion times (up to seven days)
- drugs which are excreted over prolonged periods (see appendix 3)
- factors which may delay excretion
° procedures for safe handling of body substances and soiled materials used for patient care:
- use of PPE
- procedures for containment and disposal
- special safety precautions associated with contaminated waste material from catheters,
peritoneal dialysis, colostomies etc.
3.8 Management in the community.
3.9 Record keeping.
3.10 Storage and packaging requirements (for transportation and handling).

CDC recommendations for Post Exposure to Blood Borne Pathogens

• Wounds and skin sites that have been in contact with blood or body fluids should be washed with soap and water; mucous membranes should be flushed with water.
• The person whose blood or body fluid is the source of an occupational exposure should be evaluated for HBV, HCV and HIV infection.
• Post-exposure treatment should begin within 24 hours and not later than 7 days.
• Recommendations for HBV post-exposure management include initiation of the hepatitis B vaccine series to any susceptible, unvaccinated person who sustains an occupational blood or body fluid exposure. Post-exposure prophylaxis (PEP) with hepatitis B immune globulin (HBIG) and/or hepatitis B vaccine series should be considered for occupational exposures after evaluation of the hepatitis B surface antigen status of the source and the vaccination and vaccine-response status of the exposed person.
• There is no post-exposure treatment that will prevent HCV infection. For the person exposed to an HCV-positive source perform baseline testing for anti-HCV and ALT activity; and perform follow-up testing (e.g., at 4–6 months) for anti-HCV and ALT activity (if earlier diagnosis of HCV infection is desired, testing for HCV RNA may be performed at 4–6 weeks). Immune globulin and antiviral agents (e.g., interferon with or without ribavirin) are not recommended for PEP of hepatitis
• Recommendations for HIV PEP include a basic 4-week regimen of two drugs (zidovudine and lamivudine, lamivudine and stavudine or didanosine and stavudine) for most HIV exposures and an expanded regimen that includes the addition of a third drug for HIV exposures that pose an increased risk for transmission.

Escherichia coli

ASM , CDC and FDA ahve put together a report all about E.coli and how it is transmitted. It does not have anything in it about lab testing but it is still a really good resource. Copy and paste this url:http://academy.asm.org/images/stories/documents/EColi.pdf

Prevention of Occupational Health Hazards in Hospitals

Hospital biological hazards elimination:

Elimination of biological hazards includes construction guidelines in hospitals, standard precautions, which must be applied to all patients at all times, additional (transmission-based) precautions which are specific to modes of transmission (airborne, droplet and contact), environmental cleaning, sterilization and disinfection of patient equipment (Manyele et al., 2008).
1. Construction guidelines in hospitals:
An infection control team member should participate on the planning team for any new hospital construction or renovation of existing facilities. The role of infection control in this process is to review and approve construction plans to ensure that they meet standards for minimizing nosocomial infections (Walker et al., 2007).
Considerations will usually include selection of the site of hospitals which should be away from overcrowded public areas especially for hospitals managing infectious diseases as TB, SARS, meningitis and pandemic influenza. Considerations also include proper ventilation, isolation room design, proper traffic flow and appropriate access to hand washing facilities (Lateef, 2009).


a. Ventilation:
Of all the possible engineering techniques that can be employed to control airborne pathogens, good ventilation is probably the most effective. Although most people are familiar with the general concept of ventilation, very few understand the principles on which it is based. The term ventilation should therefore only be applied to the supply of outside air to the room space (Crimi et al., 2005).
Dilution ventilation:
Most ventilation systems push large quantities of ‘clean’ outside air into occupied spaces so that any contaminants in the room space are diluted and flushed out to atmosphere. To function properly good mixing of the air in the room space is essential (McLarnon et al., 2006).
Laminar and displacement ventilation:
This is based mainly on directing airflows to displace the contaminated air so that it is ‘pushed’ out of the room space. In this way the contaminated air is continually being replaced by clean air. With this type of ventilation system it is undesirable to have any air mixing and so ‘laminar’ streams of air are often used. Such ventilation systems are frequently used in operating theatres and isolation rooms (Abdulsalam et al., 2010).





Pressure differences ventilation:
By controlling the airflows within a building it is possible to create ‘high’ and ‘low’ pressure regions. This can be used to great advantage in isolation rooms, which can be negatively pressurized space so that airborne pathogens are unable to escape. These negative pressures can be achieved by supplying less air to an isolation room than is extracted. It is recommended that a positively pressurized anteroom be placed between the corridor and the isolation room according to fire regulations (Crimi et al., 2005).
Natural ventilation:
Many hospital buildings, especially older facilities, rely heavily on natural ventilation. In many ways the natural ventilation of clinical spaces is a good solution. However, reliance solely on natural ventilation has a number of drawbacks such as ventilation rates will be variable and are greatly dependent on the outside climatic conditions. Also, Pathogens such as Aspergillus spp. which is widespread in the outdoor environment can easily enter ward spaces (McLarnon et al., 2006).
b. Room air cleaning devices:
There are a variety of room air cleaning devices currently available, incorporating technologies such as high efficiency particulate air (HEPA) filters and ultraviolet germicidal irradiation (UVGI) lamps. These devices are intended to be mounted within a room space and are designed to reduce the microbial level in the room air. They have the advantage that they are relatively cheap and can be strategically positioned to protect vulnerable patients and staff (Crutis, 2007).


High efficiency particulate air (HEPA) filters units:
HEPA filter units are readily available machines that can be used any where to provide clean air. These units are especially useful in settings that may have inadequate or no ventilation and limited funds for upgrades. HEPA filters exhibit very high efficiencies (i.e. 99.9 % efficient for particles 0.3µm in diameter). HEPA filter unit will provide cleaned air to dilute infectious particles and will also remove airborne particles. HEPA filter units are available in a variety of sizes and configurations. All consist primarily of pre-filter to remove coarser particles and thereby prolong the life of the HEPA filter, a fan to circulate the filtered into the room and controls, such as an on/off switch and fan speed control (Liao et al., 2008).
The most common types of units are portable, free standing and permanent devices. Ceiling-mounted and wall-mounted units are also available. Portable units have the advantage of greater flexibility and ease of installation and service (Eckmanns et al., 2006).
Ultra-violet (UV) germicidal irradiation:
The germicidal properties of UV light have been known about for many years; in the pre-antibiotic era. UV lamps were used extensively in tuberculosis (TB) wards to control the spread of infection, but with the development of anti-bacterial drugs and, in some countries the introduction of vaccination against TB, UV air disinfection fell out of favour. However, in recent years, with the global rise in TB, there has been renewed interest in its use as an infection control measure and several research programmes have been initiated in this field (Kujundzic et al., 2006).
The activation spectrum peaks in the range 260 to 270 nm is similar to the absorption spectrum of nucleic acids, thus deoxyribonucleic acid (DNA) is the main target. UV light at this wave length is absorbed by nucleic acids with the formation of pyrimidine dimers, resulting in damage to the DNA of the micro-organism which is lethal. Conventional low and medium pressure mercury discharge UV lamps have a strong spectral emission at 253.7 nm, close to the peak of the action spectrum and can be used as an effective bactericidal agent (Rudnick and First, 2007).
C. Isolation room design:
The interest in the use of airborne isolation rooms has increased due to the occurrence of new emerging diseases such as severe acute respiratory syndrome (SARS), avian influenza and multi-drug resistant tuberculosis (MDR-TB). The recognition of the possibility of pandemic outbreaks of influenza has required authorities to put in place “emergency planning” for dealing with infected patients that may require isolation (Walker et al., 2007).
Isolation rooms can be used to limit the spread of small-particle aerosols, which may play a role in all isolated diseases. Mechanically ventilated isolation rooms can be classified in to standard, source and protective isolation room according to the ventilation pressure as shown in table (5) (Hoffman et al., 2004).
Isolation rooms which can be switched from one functional type to another such as changing a positive-pressure room into a negative-pressure room are highly hazardous and should be avoided due to the possibility of use at the incorrect pressure regime. If multifunctional facilities are to be used staff must be trained correctly, rooms must have written operating and auditing procedures and in addition electronic warning systems and alerts must be in place (Cheong and Phau, 2006).
Isolation rooms should have minimum air-change rates of 6 air changes an hour (ach) for the protection of staff and visitors in the room. If possible, this should be increased to the 12 ach recommended as a minimum by the American institute of architects. The room airflow pattern should be designed to provide HCWs or visitors with clean air (Lateef, 2009).

d. Traffic flow:
Distinction can be made between high-traffic and low-traffic areas. A hospital with well-defined areas for specific activities can be described using flowcharts depicting the flow of in or outpatients, visitors and staff. Building or rebuilding a hospital requires consideration of all physical movements and communications and where contamination may occur (Manyele et al., 2008).
e. Architectural segregation:
It is useful to stratify patient care areas by risk of the patient population for acquisition of infection. For some units, including oncology, neonatology, intensive care and transplant units special ventilation may be desirable. Four degrees of risk may be considered; low risk areas as the administrative sections, moderate risk areas as the regular patient units, high risk areas including isolation unit and intensive care unit and very high risk areas considering the operating rooms (Tang et al., 2006).
f. Construction material:
The choice of construction material especially those considered in the covering of internal surfaces is very important. Floor coverings must be easy to clean and resistant to disinfection procedures. This also applies to all items in the patient environment (Lateef, 2009).





g. Water supply:
The physical, chemical and bacteriological characteristics of water used in healthcare institutions must meet local regulations. The institution is responsible for the quality of water once it enters the building. For specific uses, water taken from a public network must often be treated for medical use (physical or chemical treatment) (Walkera et al., 2007).
2. Standard precautions for protection of healthcare workers:
These measures must be applied during every patient care and during exposure to any potentially infected material or body fluids as blood and others. These include the following: hand washing and antisepsis (hand hygiene), use of personal protective equipment, appropriate handling of contaminated equipment and prevention of needle stick injuries (Talaat et al., 2003).
1. Hand washing:
Frequent and adequate hand washing is the best way to prevent spread of most nosocomial infections. The extreme importance of hand washing has been known since at least 1847, when Dr Ignaz Semmelweis discovered that washing hands before performing obstetric exams on pregnant women reduced childbirth-related infectious mortality from more than 10% to less than 1% (Jumaa, 2005).
For hand washing the following must be available running water, large washbasins which require little maintenance, with antisplash devices and hands-free controls, products as soap or antiseptic depending on the procedure facilities and disposable towels if possible for hand drying without contamination. For hand disinfection, specific hand disinfectants as alcohol rubs with antiseptic and emollient gels should be present (Naikoba and Hayward, 2001).
Wash of hands must be done after handling any blood, body fluids, secretions, excretions and contaminated items; between contact with different patients; between tasks and procedures on the same patient to prevent cross contamination between different body sites and immediately after removing gloves. Procedures will vary with the patient risk assessment. There are three types of hand wash as shown in the following table (2) (Jumaa, 2005).
2- Barrier precautions (Personal protective equipment):
1. Gloves:
Gloves protect the hands from contacting blood, droplets, body fluids and pathogen-contaminated objects so infection can be prevented when touching the eyes, mouth or nose afterwards. Gloves can also protect open wounds from contamination by pathogen. It is not certain what type of glove provides the best protection for infection control. Some studies have suggested that latex gloves are some what better in preventing penetration of water and virus than vinyl gloves (Wittmann et al., 2010).
2. Masks:
Using the appropriate respiratory protective equipment is important for an adequate protection from biological hazards. Surgical mask is the most common respiratory protective equipment. It should be worn in circumstances where there are likely to be splashes of blood, body fluids, secretions and excretions or when the patient has a communicable disease that is spread via the droplet route (Farrington, 2007).
N95 or higher level respirators filter out particulates and liquid droplets in small particle size, therefore providing protection from inhaling aerosols and micro-organisms that are airborne. N95 masks are used in high-risk wards (Grinshpun et al., 2009).
The mask must fit over the face with the coloured side of the mask faces outwards and the metallic strip upper most, while the strings or elastic bands are positioned properly to keep the mask firmly in place. When the mask is damaged or soiled, it is replaced. (MacIntyre et al., 2009).
If required to wear N-95 mask, the face-piece must be of proper fit. Compare the size of different brands to find a suitable one. To reuse a N95 mask, it should be kept in a paper bag properly before using it again. If the N95 mask is soiled or damaged, it must be replaced immediately. N95 mask should never be shared with any body or brought outside the hospital. N95 masks should not be used by persons suffering from respiratory diseases, such as asthma and emphysema having difficulty in breathing or feeling dizzy after wearing it at this time N95 masks with exhale valve can be used as it lets the hot air out, making the mask more comfortable (Cowling et al., 2009).
3. Protective clothing:
Protective clothing includes protective coverall (with attached hood) and gown should be water proof or impervious to liquids to protect the body from contamination by blood, droplets or other body fluids, thus reducing the chance of spreading of pathogen and cross-infection. Protective clothing is disposable in most cases though some can be reused after sterilization. Standard protective clothing should fit the wearer, checked before use and replaced if damaged. (Northington et al., 2007).
c. Goggles/Face shields:
Safety goggles/glasses and face shields can protect the eyes from contacting pathogen-carrying blood, droplets or other body fluids which may then enter the body through the mucosa. Goggles fit the face snugly and therefore are better than glasses in eye protection. If necessary, face shield should be used to protect the whole face (Davis et al., 2007).


d. Boots/shoe covers:
Boots/shoe covers are used to protect the wearer from splashes of blood, body fluids, secretions and excretions. Waterproof boots should be worn for heavily contaminated, wet flooring and floor cleaning. Shoe covers should be disposable and waterproof (Northington et al., 2007).
e. Caps:
Caps that completely cover the hair are used when splashes of blood and body fluids are expected. They should protect the hair from aerosols that may otherwise lodge on the hair and be transferred to other parts of the healthcare worker such as face or clothing by the hands or onto inanimate objects. Caps should be disposable, waterproof and of an appropriate size which completely covers the hair (Parmeggiani et al., 2010).
3. Sharp precautions:
Needle stick and sharp injuries carry the risk of bloodborne infection e.g AIDS, HCV, HBV and others. Essential elements of the prevention and control of diseases caused by sharp injuries infections include training and education about nature of bloodborne pathogens, activities that place HCW at risk and means to prevent disease transmission as personal protective procedures. Protective procedures are multifaceted and staged. Procedures include the wearing of puncture resistant gloves by all personnel in situations when blood and/or body fluids are present (Daha et al., 2009).
Needles must not be recapped after injections. Sharps should be collected at source of use in puncture-proof containers (usually made of metal or high-density plastic) with fitted covers. To discourage abuse, containers should be tamper-proof (difficult to open or break). Where plastic or metal containers are unavailable or too costly, containers made of dense cardboard are recommended. Needle incinerators can be another safe way of disposal. Reusable sharps must be handled with care avoiding direct handling during processing (Clarke et al., 2002).
CDC recommendations for post exposure to bloodborne pathogens (Parmeggiani et al., 2010):
• Wounds and skin sites that have been in contact with blood or body fluids should be washed with soap and water; mucous membranes should be flushed with water.
• The person whose blood or body fluid is the source of an occupational exposure should be evaluated for HBV, HCV and HIV infection.
• Post-exposure treatment should begin within 24 hours and not later than 7 days.
• Recommendations for HBV post-exposure management include initiation of the hepatitis B vaccine series to any susceptible, unvaccinated person who sustains an occupational blood or body fluid exposure. Post-exposure prophylaxis (PEP) with hepatitis B immune globulin (HBIG) and/or hepatitis B vaccine series should be considered for occupational exposures after evaluation of the hepatitis B surface antigen status of the source and the vaccination and vaccine-response status of the exposed person.
• There is no post-exposure treatment that will prevent HCV infection. For the person exposed to an HCV-positive source perform baseline testing for anti-HCV and ALT activity; and perform follow-up testing (e.g., at 4–6 months) for anti-HCV and ALT activity (if earlier diagnosis of HCV infection is desired, testing for HCV RNA may be performed at 4–6 weeks). Immune globulin and antiviral agents (e.g., interferon with or without ribavirin) are not recommended for PEP of hepatitis
• Recommendations for HIV PEP include a basic 4-week regimen of two drugs (zidovudine and lamivudine, lamivudine and stavudine or didanosine and stavudine) for most HIV exposures and an expanded regimen that includes the addition of a third drug for HIV exposures that pose an increased risk for transmission.
4. Handling of contaminated materials:
HCWs are exposed to contaminated equipment and working surfaces, protective coverings, reusable containers and glassware. All those should be cleaned and decontaminated after contact with blood or other potentially infectious materials with barrier precautions. Linens in a hospital can be a major potential source of infection since they are likely to be contaminated by patients in hospital beds. When linens are changed, they handled with barrier precautions and gathered in enclosed coded bags as soon as they are stripped from the bed and sent to the laundry (de Castro et al., 2009)..
On handling spots of blood or other spills, gloves and eye protection should be worn. Contamination should be wiped up with paper towels soaked in freshly prepared hypochlorite solution (milton or chlorine releasing tablets) containing 10,000 ppm (1%) available chlorine. If broken glass is present, first treat the spillage with hypochlorite and then carefully remove the pieces of glass with disposable forceps or scoop to a sharps bin, before wiping up. Towels and gloves should be disposed of in a yellow clinical waste bag for incineration (or an autoclave bag if in a laboratory). Hands must be washed following clearing up. Spilt blood should not be allowed to dry as potential aerosol production is greater from dried blood (CDC, 2004).

3. Transmission route based precautions:
a. Contact Precautions
In addition to standard precautions, contact precautions are indicated for patients known or suspected to have serious illnesses easily transmitted by direct patient contact or by contact with items in the patient’s environment. In addition to standard precautions, contact precautions include putting on PPE (such as gowns) prior to entry into a patient room and taking off PPE prior to exit. Patient care equipment must be dedicated with limitation of patient movement. Those patients must be placed in a private room or with patients who have active infection with the same micro-organism with no other infection (Curtis, 2008).
b. Droplet Precautions
Droplet precautions are indicated for patients known or suspected to have serious illnesses transmitted by large particle droplets, such as seasonal influenza, invasive haemophilus influenzae type b disease and invasive neisseria meningitidis. In addition to standard precautions, droplet precautions include the use of a surgical mask when working within 3 feet of the patient and the placement of the patient in a private room or with patients who have an active infection with the same micro-organism but with no other infection (Farrington, 2007).






c. Airborne Precautions
Airborne Precautions are designed to reduce the risk of airborne transmission of infectious agents as H5N1 avian influenza, SARS, measles, varicella and tuberculosis. In addition to standard precautions, airborne precautions include placement of the patient in a negative pressure room (airborne infection isolation room), if available. If a negative pressure room is not available or cannot be created with mechanical manipulation of the air, place patient in a single room. Doors to any room or area housing patients must be kept closed when not being used for entry or egress. When possible, isolation rooms should have their own hand washing sink, toilet and bath facilities (Tormo et al., 2002).
The number of persons entering the isolation room should be limited to the minimum number necessary for patient care and support. CDC recommends the use of a particulate respirator that is at least as protective as NIOSH certified N95. For patients for whom influenza is suspected or diagnosed, surveillance, vaccination, antiviral agents and use of private rooms as much as feasible is recommended. In contrast to tuberculosis, measles and varicella, the pattern of disease spread for seasonal influenza does not suggest transmission across long distances (e.g., through ventilation systems); therefore, negative pressure rooms are not needed for patients with seasonal influenza (Lateef, 2009).

4- Environmental cleaning:
Cleaning is the removal of all visible and invisible organic material (e.g., soil) from objects to prevent micro-organisms from thriving, multiplying and spreading. 90% of micro-organisms are present within “visible dirt” and the purpose of routine cleaning is to eliminate this dirt. Neither soap nor detergents have antimicrobial activity and the cleaning process depends essentially on mechanical action (Nchez-Paya et al., 2009).
There must be policies specifying the frequency of cleaning and cleaning agents used for walls, floors, windows, beds, curtains, screens, fixtures, furniture, toilets and all reused medical devices. Methods must be appropriate for the likelihood of contamination and necessary level of asepsis.
5. Disinfection of patient equipment
Disinfection removes micro-organisms without complete sterilization to prevent transmission of organisms between patients. Disinfectants must be easy to use, non-volatile and not harmful to equipment, staff or patients, free from unpleasant smells and effective within a relatively short time. Different products or processes achieve different levels of disinfection. These are classified as high, intermediate or low level disinfection. Table (8) provides characteristics of the three levels (Hedin et al., 2010).

Monday, December 5, 2011

Community-Acquired Pneumonia (CAP)

Community-acquired pneumonia (CAP) is a common health problem that may still be life threatening in an age of wide availability of effective antibiotic therapy. The annual incidence rate rises from 6/1000 in the 18–39 age group to 34/1000 in people aged 75 years and over (Blasi et.al., 2007). Hospital admission is necessary in 20–40% of cases, with 5–10% of these patients being admitted to an intensive care unit (ICU). Overall mortality from CAP is 5–10% (Hoare and Lim, 2006).

Bacterial infection
Streptococcus pneumonia
Haemophilus influenzae
Escherichia Coli
Klebsiella pneumoniae
Pseudomonas aeruginosa
Atypical infection
Mycoplasma pneumoniae
Coxiella burnetti
Chlamydia psittaci
Legionella pneumophila
Fungal infection
Aspergillus
Candida
Nocardia
Histoplasmosis

Viral infections
Influenza
Coxsackie
Respiratory syncytial
Cytomegalovirus
Adenovirus Protozoal infections
Pneumocystis carinii
Toxoplasmosis
Amoeboesis Others
Aspiration
Bronchiectasis
Cystic fibrosis
Lipoid pneumonia
Radiation

Saturday, December 3, 2011

Microbiological Classification of Pneumonia

Pneumonia classified according to the probable origin of infection into community acquired pneumonia or nosocomial (hospital acquired) pneumonia (Wilkinson and Woodhead, 2004).

a) Community acquired pneumonia
Community-acquired pneumonia (CAP) is defined as an infection of the alveolar or gas-exchanging portions of the lungs occurring outside the hospital, with clinical symptoms accompanied by the presence of an infiltrate in the chest radiograph (Lippi et.al., 2011).
Most bacterial infections result from aspiration of nasopharyngeal organisms, although inhalation of infected droplets from other patients, from animals, or from environmental sources , less commonly, hematogenous spread to the lung (Baltzer et.al., 2006).

b) Nosocomial pneumonia
The spectrum of nosocomial pneumonia can include hospital acquired pneumonia (HAP), health care associated pneumonia (HCAP), and ventilator associated pneumonia (VAP) (Jevtic, 2009).

Hospital acquired pneumonia is defined of pneumonia occuring at least 48 hours after hospital admission (Kollef and Micek, 2005).
It may result as a direct result of surgery (postoperative and aspiration pneumonia) but is more often a result of major co morbidity, for example chronic obstructive pulmonary disease (COPD) or general debility in elderly. Two to five percent of hospital admission are complicated by development of this condition (Yarnell, 2007).
Ventilator associated pneumonia is defined as nosocomial Pneumonia in patients receiving mechanical ventilation via an endotracheal tube that develops 48 hours or more after initiation of ventilation ( curley et. al., 2006).
Ventilator associated pneumonia is the most frequent infection in intensive care
units (ICU) and has a high mortality and morbidity rate (Bouza et.al., 2011). It occurs in 8% to 28% of patient receiving mechanical ventilation in ICU (Wu et.al., 2011).
Heath care associated pneumonia [HCAP] :- This category includes patients who a] receive home nursing, intravenous antibiotics, or wound care; b] patients who reside in nursing homes or long-term care facilities; c] patients who have been hospitalized for ≥ 2 days in the past 90 days; d] patients who have received dialysis or IV therapy at a hospital-based clinic in the past 30 days (Kronish and Dorr, 2008).

Wednesday, November 30, 2011

Egypt Ellection

Iam really very happy for the ellection in Egypt. Iam near fivty years old and I did not see free ellection in my country before. Sorry Ican not now write in microbiology as my mind is really occupied. Iwill writ again, but now it is the time of my country.

Tuesday, November 29, 2011

Egypt Today

Congratulations for Egyptian

Monday, November 28, 2011

Pneumonia

Pneumonia is an important cause of morbidity and mortality in adults and children. It results from the host inflammatory response to infection of the distal airways and the lung alveoli (Schutter et.al, 2011). The most useful classification is based on the site of acquisition: community-acquired (CAP) or hospital-acquired pneumonia (HAP) (Esperatti and Marti, 2008).
Hospital-associated pneumonia (HAP) is the second most common nosocomial infection (after urinary tract infection) and the most common nosocomial infection acquired in the intensive care unit (ICU). HAP associated with mechanical ventilation is called ventilator-associated pneumonia (VAP) (Franzetti et.al., 2010). VAP has an estimated incidence of 8–28% and is associated with an excess of ICU stay, increased costs, and attributable mortality (Safdar et.al., 2005).
The term atypical pneumonia was originally used to describe an unusual presentation of pneumonia. It is now more widely used in reference to either pneumonia caused by a relatively common group of pathogens, or to a distinct clinical syndrome the existence of which is difficult to demonstrate (Murdoch and Chambers, 2009).
The “atypical pathogen” group includes Mycoplasma pneumoniae; Legionella; Chlamydia pneumoniae; Coxiella burnettii; and the respiratory viruses, especially
Introduction and Aim of the Work
influenza A and B, parainfluenza 1, 2, and 3, respiratory syncytial virus (RSV) and Epstein-Barr virus (EBV) (Lieberman, 2005).
The diagnosis of pneumonia in the hospitalized patient is even more challenging than the diagnosis of CAP (Richards et.al., 2000). Clinical findings alone are not sufficient for a definitive diagnosis. Therfore, a variety of noninvasive and invasive tests have been proposed as guides for diagnosis and treatment of hospital-acquired pneumonia. Methods include sputum Gram stain and culture, serologic studies, antigen detection tests, and nucleic acid amplification methods (Carroll, 2002).
Many patient- and disease-specific factors contribute to the pathophysiology of HAP, particularly in the surgical population. Risk-factor modification and inpatient prevention strategies can have a significant impact on the incidence of HAP (Kieninger and Lipsett, 2009).

Pneumonia

Pneumonia is an important cause of morbidity and mortality in adults and children. It results from the host inflammatory response to infection of the distal airways and the lung alveoli (Schutter et.al, 2011). The most useful classification is based on the site of acquisition: community-acquired (CAP) or hospital-acquired pneumonia (HAP) (Esperatti and Marti, 2008).
Hospital-associated pneumonia (HAP) is the second most common nosocomial infection (after urinary tract infection) and the most common nosocomial infection acquired in the intensive care unit (ICU). HAP associated with mechanical ventilation is called ventilator-associated pneumonia (VAP) (Franzetti et.al., 2010). VAP has an estimated incidence of 8–28% and is associated with an excess of ICU stay, increased costs, and attributable mortality (Safdar et.al., 2005).
The term atypical pneumonia was originally used to describe an unusual presentation of pneumonia. It is now more widely used in reference to either pneumonia caused by a relatively common group of pathogens, or to a distinct clinical syndrome the existence of which is difficult to demonstrate (Murdoch and Chambers, 2009).
The “atypical pathogen” group includes Mycoplasma pneumoniae; Legionella; Chlamydia pneumoniae; Coxiella burnettii; and the respiratory viruses, especially
Introduction and Aim of the Work
influenza A and B, parainfluenza 1, 2, and 3, respiratory syncytial virus (RSV) and Epstein-Barr virus (EBV) (Lieberman, 2005).
The diagnosis of pneumonia in the hospitalized patient is even more challenging than the diagnosis of CAP (Richards et.al., 2000). Clinical findings alone are not sufficient for a definitive diagnosis. Therfore, a variety of noninvasive and invasive tests have been proposed as guides for diagnosis and treatment of hospital-acquired pneumonia. Methods include sputum Gram stain and culture, serologic studies, antigen detection tests, and nucleic acid amplification methods (Carroll, 2002).
Many patient- and disease-specific factors contribute to the pathophysiology of HAP, particularly in the surgical population. Risk-factor modification and inpatient prevention strategies can have a significant impact on the incidence of HAP (Kieninger and Lipsett, 2009).

Saturday, November 26, 2011

Innate Immune Response to Pathogens and Recent Advances in Microbiology Researches

An immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own healthy cells and tissues in order to function properly. Detection is complicated as pathogens can evolve rapidly, and adapt to avoid the immune system and allow the pathogens to successfully infect their hosts.
To survive this challenge, multiple mechanisms evolved that recognize and neutralize pathogens. Even simple unicellular organisms such as bacteria possess enzyme systems that protect against viral infections. Other basic immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants, such as plants and insects. These mechanisms include antimicrobial peptides called defensins, phagocytosis, and the complement system. Jawed vertebrates, including humans, have even more sophisticated defense mechanisms. The typical vertebrate immune system consists of many types of proteins, cells, organs, and tissues that interact in an elaborate and dynamic network. As part of this more complex immune response, the human immune system adapts over time to recognize specific pathogens more efficiently. This adaptation process is referred to as "adaptive immunity" or "acquired immunity" and creates immunological memory. The following chapters will discuss various mechanisms of immune response to pathogens based on laboratory evidence studies.







Friday, November 25, 2011

Qualitative and quantitative detection of endotoxin in serum

Endotoxins are large (molecular weight, 200,000 to 1,000,000), heat-stable lipopolysaccharides (LPS) which are the major components of the cell wall of the gram-negative bacterium.There are more than 20 assays for the detection of endotoxin (McCabe, W. R., 1980), of which three have been used for the detection of endotoxin in clinical specimens: the rabbit pyrogen assay, the LAL

bioassay, and immunoassays. The method of choice would appear to be the LAL assay. The advantages of this assay are increased sensitivity, potential for quantitation, reactivity with the biologically active component lipid A, and relative convenience of operation.

The LAL Assay

In 1956, Bang (Bang, 1956) discovered that the endotoxin of a Vibrio species from seawater, pathogenic for the horseshoe crab (Limulus polyphemus), caused fatal intravascular coagulation and that endotoxin induced activation of this process in vitro. Levin, Bang, and coworkers subsequently showed that this coagulation was the result of an endotoxin-initiated reaction causing the enzymatic conversion of a clottable protein derived from the circulating blood cell (amebocyte) of the crab (Levin, et al.,1968; Young, et al., 1972). They recognized the potential for this biological reagent as a diagnostic tool and characterized its properties. A lysate from the amebocyte is extremely sensitive to the presence of endotoxin.

Coagulation system of L. polyphemus.

The coagulation system of L. polyphemus consists of several enzymes which are arranged in three pathways in a fashion which resembles the classic, alternate, and common mammalian coagulation cascade pathways, the components of which activate each other in a ‘‘cascade’’ sequence. The coagulation system of the Japanese horseshoe crab, T. tridentatus, which is considered homologous to the L. polyphemus American horseshoe crab, has been studied extensively (Fig.3) (Iwanaga, S., 1993; Iwanaga, et al., 1985). This

cascade sequence results in an amplification of the original stimulus which accounts for the sensitivity of the Limulus coagulation system to endotoxin at picogram-per-milliliter (10-12 g/ml) concentrations. An additional component of Limulus amebocytes is an anti-LPS factor which has anti-endotoxin properties (Warren, et al., 1992).
Gel clot LAL assay.
In the original version of the gel clot test, the endotoxin-activated clotting enzyme cleaves the coagulogen to form a clot. To perform this test, a small amount of LAL solution is added to an equal volume of a sample or a standard dilution in a small test tube. If, after an appropriate incubation time, a firm gel clot is formed, the test is scored positive. A firm gel clot is one that remains solid in the bottom of the reaction tube when the tube is inverted. Methods to

enhance the visualization of clot formation in microtiter volumes have been described (Gardi, et al, 1980; Hussaini, et al., 1987; Prior, et al., 1979). With all gel clotbased techniques, a semiquantitative result can be obtained through serial dilution of samples and standards.

Coagulogen-based LAL assay.
The limitations of the gel clot LAL test are the subjective endpoint and the relative lack of sensitivity. To overcome these limitations, various methods to quantitate the progress of the reaction leading to coagulogen conversion have been employed, for example, through monitoring the increase in turbidity (Dubczak, et al., 1979; Urbaschek, et al., 1985), the loss of coagulogen as the clot forms (Baek, 1983; Zhang, et al., 1988), the increase in precipitated protein (Nandan, et al., 1977; Nandan, et al, 1977), or the appearance of a peptide cleavage fragment of coagulogen (Zhang, et al., 1994).

Chromogenic LAL assay.
In the chromogenic LAL assay method (Iwanga, et al ., 1978), the coagulogen is completely or partially removed to be replaced by a chromogenic substrate (Scully, et al., 1980), a small synthetic peptide linked to a chromophore (para-nitroaniline) containing an amino acid sequence similar to that present at the site in the clotting protein cleaved by the clotting enzyme (X-Y-Gly-Arg-pNA). The chromogenic LAL assay usually has two stages: a LAL activation stage and, following the addition of the chromogenic substrate to the reaction mixture, a chromophore release stage. Release of the chromophore imparts a yellow color to the solution. The strength of the yellow color (as measured by optical density [OD] at 405 nm in a spectrophotometer) is a function of the amount of active clotting enzyme (and indirectly to the amount of endotoxin)

present in the solution. Both phases of the chromogenic reaction are critically time and temperature dependent, but within these limitations the chromogenic assay is sensitive to 10 pg/ml (Thomas, et al., 1981). A single-step chromogenic assay has been described (Duner, K. I., 1993; Lindsay, et al., 1989).

Specificity of the LAL Assay

The two pathways leading to the coagulation of LAL, one activated by endotoxin triggered by factor C and the other activated by b-glucans triggered by a glucan-reactive factor G, can be specifically blocked by polymyxin and laminarin, respectively (Zhang, et al., 1994). Hence, reactivity with the LAL assay that is inhibited by polymyxin B can be used as specific evidence for endotoxin. LAL derived from the Japanese horseshoe crab and from which this factor G has been removed has been promoted as an endotoxin-specific reagent (Obayashi, et al., 1985, Obayashi, et al., 1986).

LAL Endotoxin Assay for Blood Samples

When the LAL assay is used to detect endotoxin in blood, two obstacles are encountered: (i) the complex and poorly understood inhibitory factors and (ii) the levels of endotoxemia generally being at the limit of test detection. Schematic overview 1. illustrates the complex interaction among components of blood, endotoxin, and LAL. Endotoxin interacts with several components of plasma, including bile salts, proteins, and lipoproteins, leading to disaggregation, some inactivation, and the formation of complexes. These multiple effects of plasma on the activity of endotoxin are not always apparent as inactivation. (Beller, et al., 1963).


Inhibition by plasma and serum.
The loss of reactivity to LAL on addition of endotoxin to plasma or serum is partly reversible, in that reactivity can be restored by dilution with distilled water or saline (Levin, et al., 1970), and partly irreversible (Johnson, et al., 1977). The ability of plasma or serum to inhibit endotoxin activity is time dependent and temperature sensitive, being maximal at 37 to 45°C and abolished after plasma or serum is heated at 60°C for 5 min, and varies in proportion to the endotoxin potency. These characteristics imply an enzymatic inactivation of endotoxin by native plasma (Johnson, et al., 1977; Novitsky, et al., 1985, Novitsky, et al., 1985, Obayashi, T., 1984, Olofsson, et al., 1986, Webster, et al., 1980), although this has yet to be definitively demonstrated.

Endotoxemia without Sepsis
Liver disease. Endotoxemia has been suspected of having pathogenic properties in patients with liver disease even in the absence of overt gram-negative sepsis (Nolan, J. P., 1975). The origin of endotoxin in this setting is also believed to be from the gastrointestinal tract because several studies have found a portal-to-systemic gradient of endotoxin level, with higher level in portal venous blood than in peripheral blood ( Bigatello, et al, 1987; Jacob, et al, 1977; Lumsden, et al, 1988; Prytz, et al, 1976).

Hemodialysis. pyrogenic reactions are an important problemwith hemodialysis, and there is concern that this is due to contamination of the dialysis water with bacteria or endotoxin (Pegues, et al, 1992; Raij, et al, 1973) or contamination resulting from the use of reprocessed dialyzers (Flaherty, et al, 1993; Gordon, et al, 1988). There is uncertainty as to whether endotoxin is able to cross the different types of dialyzer membranes and also whether the LAL-

reactive material (LAL-RM) found in the plasma of patients undergoing hemodialysis is something other than endotoxin. It is suggested that the LAL-RM is a cellulose-based material, possibly (1-3)-β-D-glucans, which has properties distinct from endotoxin (Roslansky, et al, 1991) and reacts with the factor G-drive pathway of LAL (Zhang, et al,1994). An endotoxin specific assay which does not react with (1-3)- β-D-glucans has been developed and applied (Taniguchi, et al, 1990). In any event, LAL testing of plasma of hemodialysis patients has limited ability to detect pyrogenic reactions, having positive and negative predictive values of less than 70% (Gordon, et al, 1992).

Intestinal endotoxemia. An origin from the gastrointestinal tract has often been presumed for endotoxemia in patients with gastrointestinal diseases (Cooperstock, et al, 1985; Wellmann, et al, 1984) and also in patients receiving radiotherapy to the abdomen in association with symptoms of nausea (Maxwell, et al, 1986).

Other conditions. Transient endotoxemia occurs in patients undergoing minimally invasive procedures of the urinary (Garibaldi, et al, 1973; Robinson, et al, 1975; Tanaka, et al, 1988), biliary (Lumsden, et al, 1989), or gastrointestinal (Kelley, et al, 1985) tract. In general, the severity of symptoms and the degree or frequency of detection of endotoxemia in these patients are higher when gram-negative bacteria are found at the sites of these procedures. In premature neonates, there is an association between endotoxin in cord blood and growth of gram-negative bacteria from placental samples (Scheifele, et al, 1984).

Thursday, November 24, 2011

Cellular and Humoral Response Involved in Gram-Negative Bacterial Sepsis and Septic Shock

As soon as a bacterium enters the body, it is confronted with two lines of defense: a humoral line and a cellular line. The humoral factors comprise complement, antibodies, and acute phase proteins. In the cellular line of defense, in particular the mononuclear cells (monocytes and macrophages) and the neutrophils are of great significance since these cells may recognize bacterial cell wall constituents directly or indirectly after complement and antibody bind to the bacterium and its constituents. It is now thought that continuous challenges with small amounts of bacterial constituents may be necessary to keep the immune system alert to infections. Indeed, low levels of LPS are present in healthy individuals without causing disease (Takakuwa, et al., 1994; Vogel, et al., 1990).

Lipopolysaccharide

While the terms endotoxin and LPS are used interchangeably, the former term to emphasize the biological activity and the latter term to refer particularly to the chemical structure and composition of the molecule (Hitchcock, et al., 1986; Qureshi, et al., 1991). LPS is a major constituent of the outer membrane of gram-negative bacteria and is the only lipid constituent of the outer leaflet (Rietschel, et al., 1994). LPS is an essential compound of the cell wall and is a

prerequisite for bacterial viability. It is not a toxic molecule when it is incorporated into the bacterial outer membrane, but after release from the bacterial wall, its toxic moiety, lipid A, is exposed to immune cells, thus evoking an inflammatory response. LPS and other cell wall constituents are released from the bacterial cells when they multiply but also when bacteria die or lyse (Hellman, et al., 2000, Rietschel, et al., 1994). Various endogenous factors like complement and bactericidal proteins can cause disintegration of bacteria, resulting in the release of LPS (De Bleser, et al., 1994). In addition, some antibiotics are known to cause the release of LPS from bacteria (Crosby, et al., 1994).

The LPS molecule consists of four different parts (Fig1,2). (Lugtenberg, et al., 1983; Raetz, et al., 1990; Rietschel, et al., 1994).The first and most essential part is lipid A, the covalently linked lipid component of LPS. Six or more fatty acid residues are linked to two phosphorylated glucosamine sugars. All bacterial species carry unique LPS. Experiments with synthetic lipid A have shown that this part of the LPS molecule represents the toxic moiety (Kotani, et al 1985). The second part of the LPS molecule is the inner core, which consists of two or more 2-keto-3-deoxyoctonic acid (KDO) sugars linked to the lipid A glucosamine and two or three heptose (L-glycero-D-manno-heptose) sugars linked to the KDO. Both sugars are unique to bacteria. The outer core, the third part of the LPS molecule, consists of common sugars and is more variable than the inner core. It is normally three sugars long with one or more covalently bound sugars as side chains. LPS serotypes consisting of lipid A and the complete inner and outer core are denoted Ra-LPS, whereas the Rb- and Rc-LPS serotypes only contain a part of the outer core. The fourth moiety of the LPS molecule is the O antigen. This part of the LPS molecule is attached to the

terminal sugar of the outer core, extends from the bacterial surface, and is highly immunogenic. It is composed of units of common sugars, but there is a huge interspecies and interstrain variation in the composition and length (Edwin S, et al., 2003).
Cellular defense:

LPS and other bacterial (surface) components are recognized by complement and antibodies, leading to opsonisation and lysis of the bacterium. Phagocytes (monocytes, macrophages, and polymorphonuclear leukocytes [PMN]) are able to recognize opsonized bacterial components by complement receptors and Fc receptors (which bind immunoglobulin G [IgG] antibodies) (Frank, et al., 1991).

In the host response to bacteria, the mononuclear phagocytes (monocytes and macrophages) are of major importance. Recognition of LPS or other bacterial components by these cells initiates a cascade of release of inflammatory mediators, vascular and physiological changes, and recruitment of immune cells. An LPS-activated macrophage becomes metabolically active and produces intracellular stores of oxygen free radicals and other microbicidal agents (lysozyme, cationic proteins, acid hydrolases, and lactoferrin) and secretes inflammatory mediators (Hiemstra, et al., 1993; Mayer, et al., 1991; Roitt, I. M., 1994). One of the key mediators is TNF-α which is one of the first cytokines released by macrophages (Beutler, et al., 1985). The release of TNF-α,

IL-1, IL-6, IL-8, IL-12, platelet-activating factor (PAF), chemokines, and eicosanoids has profound effects on the surrounding tissue (Hack, et al., 1997; Katori, et al., 2000; Lukacs, et al., 1999).

The extravasation of PMN is enabled by vasodilatation and upregulation of adhesion molecules on endothelial cells, PMN, and macrophages (Jaeschke, H., and C. W. Smith., 1997; Kawamura, et al., 1995; Van Oosten, et al., 1995). The PMN react to these stimuli by intravascular aggregation, adherence to the endothelium, diapedesis, and the production of inflammatory mediators like TNF-α, leukotriene B4, and PAF (Mulligan, et al., 1993; Van Epps, et al., 1993). The (activated) PMN express CD14, CD11/CD18, and several complement and Fc receptors and are thus able to recognize and phagocytose LPS, bacterial fragments, and whole bacteria. As specialized phagocytes, PMN produce an impressive series of microbicidal agents, such as lysozyme, bactericidal/permeability increasing protein (BPI), enzymes, and oxygen free radicals (Chatham, et al., 1993; Roitt, I. M., 1994). These agents are used mainly for lysosomal killing of microorganisms. However, adherence of the PMN to endothelial cells and the presence of high concentrations of stimuli may also result in the release of microbicidal agents; much of the endothelial damage observed in sepsis is caused by these agents (Bone, et al., 1991). Endothelial cells respond to LPS (via soluble CD14) and to the circulating cytokines by the release of IL-1, IL-6, eicosanoids, the vasoactive agents endothelium derived relaxation factor, endothelin-1, chemokines, and colony stimulating factors (CSF) (Mahalingam, et al., 1999).

The inflammatory mediators secreted by the different cell populations attract and activate B and T lymphocytes. In turn, the latter release mediators such as

IL-2, gamma interferon (IFN-γ), and granulocyte-macrophage (GM)-CSF. IL-2 and GM-CSF are involved in proliferation and activation of PMN and mononuclear cells, whereas IFN-γ enhances the effects of LPS on mononuclear cells (Bone, R. C., 1991; Heinzel, et al., 1994; Jaeschke, H., 1996; Ying, et al.,1993). The actions of the activated immune cells combined with the effects of the inflammatory mediators cause symptoms such as fever, endothelial damage, capillary leakage, peripheral vascular dilatation, coagulation disorders, microthrombi, and myocardial depression. These phenomena may finally result in multiple organ dysfunction, shock, and death (Bone, et al., 1991).

Humeral response

Bacteria activate both complement pathways: i) alternative pathway which is triggered by binding polysaccharide surface components (O antigen, capsule, and LPS) to complement factor 3 (C3) (Joiner, et al., 1984; Quezado, et al., 1994; Tesh, et al., 1988) ii) classical pathway which is activated by binding Lipid A to C1q (Ying, et al., 1993). The classical complement pathway is also activated in the presence of specific antibodies (IgG and IgM) against gram-negative bacterial constituents. In all three cases, C3b is deposited on the molecule or cell surface, which promotes phagocytosis by macrophages and neutrophils and leads to insertion of C5–C9 (membrane attack complex) into the cell surface, leading to lysis of the bacterium (De Boer, et al., 1993; Frank, et al., 1991). However, long O-antigen chains in gram-negative bacteria may protect the bacteria from complement-mediated lysis (Haeney, M. R., 1998). With the cleavage of C3 and C5, the chemoattractive and vasoactive agents C3a and C5a are released. They cause increased vascular permeability, upregulate adhesion molecule expression on endothelial cells and neutrophils, and attract and activate

these phagocytes. Furthermore, they activate basophilic granulocytes and mast cells: these cells release a variety of vasoactive compounds (such ashistamine), facilitating the invasion of phagocytes. (Espevik, et al., 1993; Hsueh, et al., 1990; Kuipers, et al., 1994; Mulligan, et al., 1993; Pu¨schel, et al., 1993; Roitt, I. M., 1994; Tesh, et al., 1988; Van Epps, et al., 1993)

During infection, liver parenchymal cells are stimulated by TNF-α, IL-1, and IL-6 to produce acute-phase proteins. These proteins comprise C-reactive protein, serum amyloid A, lipopolysaccharide- binding protein (LBP), serum amyloid P, hemopexin, haptoglobin, complement C3 and C9, α1-acid glycoprotein, α2-macroglobulin, and some proteinase inhibitors (476, 498). The expression is differentially upregulated from several fold (C3 and C9) to even 1,000-fold (C-reactive protein). Some of the acute-phase proteins, like LBP modulate the immune response reactions by activation of phagocytes and antigen-presenting cells, but basically the acute-phase response is considered to alleviate the damage caused during infection (Fey, et al., 1994; Kuipers, et al., 1994; Ramadori, et al., 1990). Albumin is a so-called negative acute-phase protein since its production is down regulated during inflammation (Fey, et al., 1994).

Tuesday, November 22, 2011

Nucleic Acid Analysis Without Amplification

It is molecular methods not based on the amplification of the target have been used.
Nucleic acid analysis without Amplification (Nucleic acid probe technology)
A nucleic acid probe is a labeled sequence of single stranded DNA or RNA that can hybridise specifically with its complementary sequence (Smith, 2002).
Nucleic acid hybridization technique
Fluorescent in situ hybridization (FISH) is a tool that today is widely used for identification, visualization and localization of microorganisms in many fields of microbiology
FISH was mainly applied in connection with environmental samples during the first years but it became clear that the method also has advantages in diagnostic microbiology for rapid identification and direct visualization of bacteria
Nucleic hybridization refer to formation of hydrogen bonds between nucleotides of single stranded DNA and/or RNA molecules that are complementary to each other. This form a stable double stranded nucleic acid molecule. The resulting double stranded hybrids may be DNA:DNA; DNA:RNA, or RNA:RNA.
This hybridization process called duplex formation. This process is key of component for many testes including blotting methods, PCR, and other molecular based techniques.
The two single stranded nucleic acid molecules used in hybridization techniques are referred to by different terms. One of the strands is known as the target.
The target strand is the DNA or RNA sequence that will be identified by the employed molecular diagnostics method. The target is referred to as the template it can be either immobilized on a solid support mechanism or is suspended in solution.The other strand is called the probe. The probe is usually a single stranded DNA or RNA oligonucleotide that is labeled with an attached reporter chemical or a radionucleotide that can be detected either visually, by film, or by an instrument. The probe is produced synthetically to detect a specific target.
Hybridization reaction variables
Several variables affect the outcome of a given hybridization reaction. These variable include;
-Temperature
The stability of a given hybrid can be calculated by determining the melting temperature (™) of a probe. The Tm is the temperature at which 50% of hybrids have formed and 50% of the single stranded nucleic acid molecules are still dissociated. Tm is dependent on the G+C ratio because three hydrogen bonds form between G and C, instead of the two hydrogen bonds that form between adenine (A) and (T);The G\C bond pair is more thermodynamically stable than the A\T bond pair.
-Length of the probe
Another aspect that affects the Tm is the length of the probe ; in general the Tm is lower for a shorter probe.Hybridization reactions tend to occur more rapidly for shorter probes than for longer probes
-Probe Concentration
Higher probe concentrations typically lower the reaction time by saturating all of the available probe target sequences. However, excessive probe concentrations promote nonspecific binding of the probe to non target sequences
-Salt concentration
The rate of a hybridization reaction will increase as the salt concentration increases, up to a threshold; past 1.2 M Nacl, the rate of the reaction become constant.
-PH
Neutral PH is preferable for most hybridization reactions
-Probe selection
Selection of proper probe for nucleic acid hybridization reaction is important as the hybridization method itself. probes may be either DNA or RNA based, and are either radiolabeled or non isotopically labeled.
Radiolabeled probes are rarely used due to have short half lives and un desirable waste.radiolabeled probes have been replaced by nonisotopic labels, including biotin digoxigenin (DIG), and fluorescein, nonisotopic labels have resolution and sensitivity that approache .A probe may also be end labeled or continuously labeled
Hybridization Formats
Hybridization reaction may occur in a solid support mechanism, in situ or in solution
A-Solid support hybridization
Technique often called (blotting) the target nucleic acid is transferred and immobilized to a membrane, composed of either nitrocellulose or nylon. Labeled probe is then hybridized to the immobilized nucleic acid washing steps are used to remove excess probe. Two examples of solid support hybridization techniques are:
1-Southern Blot
The southern blot was first described in 1975 by E.M southern; he described a technique whereby chromosomal DNA has digested with a restriction enzyme then separated by agarose gel electrophoresis, then transferred and immobilized to a nitro cellulose membrane, then labeled probe is hybridized to the specific target DNA sequences. [Figure, 5]The southern blot takes more than one day to perform, it can be used to identified micro organism, to detect mutation, to type strains for epidemiological investigation, and for other purposes (Vanrompay.d., 2000)
2- Northern Blot
A northern blot was first described by Alwine et al 1977; northern blot used to determine the size of particular RNA transcript. It has the same procedure of the southern blot but with difference, the restriction enzyme is not used to digest RNA before separation due to RNA is small enough to be separated by agaros gel electrophoresis. Like southern blotting, northern blotting is not often used in clinical microbiology laboratories
3-In Situ Hybridization
In Situ Hybridization (ISH). This is a method of hybridization wherein DNA or RNA transcript can be detected directly in the tissue with labeled probes.ISH may used to detected low level of viruses in tissue specimen such as human papillomaviruses (HPV)
An example is fluorescence in situ hybridization (FISH) with oligonucleotide probes targeting bacterial or fungal genes (typically rRNA genes) As conventional FISH probes, they are usually designed to target naturally abundant rRNA genes, thereby allowing the detection of microorganisms without the need for an amplification step .
An evolution of classical oligonucleotide probes are peptide nucleic acid (PNA) probes, which are synthetic oligomers mimicking the DNA or the RNA structure. In PNA probes, the negatively charged (deoxy)ribose-phosphate nucleic acid backbone is replaced by an uncharged N-(2-aminoethyl)-glycine scaffold to which the nucleotide bases are attached via a methylene carbonyl linker (151). Due to their neutral charge, PNA probes have more robust hybridization characteristics than those of DNA probes. As conventional FISH probes, they are usually designed to target naturally abundant rRNA genes, thereby allowing the detection of microorganisms without the need for an amplification step
Finally, and adding to their clinical applicability, PNA-FISH probes are less susceptible to inhibition by impurities in different clinical samples than amplified NAT-based methods
B-In Solution Hybridization
In solution hybridization is the type of hybridization reaction most often used by clinical microbiology laboratories.Hybridization between a labeled probe and target nucleic acids in a liquid solution in tubes or in microtiter wells, usually detection methods are chemiluminescent based. It used to rapidly identify infectious disease organisms.

Monday, November 21, 2011

Ribotyping in clincal microbiology

Ribotyping involves the fingerprinting of genomic DNA restriction fragments that contain all or part of the genes coding for the 16S and 23SrRNA. Conceptually, ribotyping is similar to probing restriction fragments of chromosomal DNA with cloned probes (randomly cloned probes or probes derived from a specific coding sequence such as that of a virulence factor).
Ribotyping assays have been used to differentiate bacterial strains in different serotypes and to determine the serotype(s)most frequently involved in outbreaks . This technique is especially useful in epidemiological studies for organisms with multiple ribosomal operons, such as members of the family of Enterobacteriaceae. Ribotyping simplifies the microrestriction patterns by rendering visible only the DNA fragments containing part or all of the ribosomal genes. The technique is less helpful when the bacterial species under investigation contains only one or a few ribosomal operons. In these instances, ribotyping typically detects only one or two bands, which limits its utility for epidemiological studies. Most studies have indicated that PFGE is superior to ribotyping for analysis of common nosocomial pathogens.

Saturday, November 19, 2011

Restriction Enzyme Pattern in Clinical Microbiology

Restriction endonucleases recognize specific nucleotide sequences inDNA and produce double-stranded cleavages that break the DNAinto small fragments. The number and sizes of the restrictionfragments, called restriction fragment length polymorphisms (RFLPs) , generated by digesting microbial DNA are influenced by both therecognition sequence of the enzyme and the composition of theDNA. In conventional restriction endonuclease analysis, chromosomalor plasmid DNA is extracted from microbial specimens and thendigested with endonucleases into small fragments. These fragmentsare then separated by size with use of agarose gel electrophoresis.The nucleic acid electrophoretic pattern can then be visualizedby ethidium bromide staining and examination under UV light(Lodish et al., 2004).
Restriction endonuclease analysis has the advantage of being highly reproducible, very accurate in determining the relatedness of microbial strains, and well within the technical capabilitiesof experienced laboratory technologists. However, the majorlimitation of this technique, especially for chromosomal DNA,is the difficulty of comparing the complex profiles generated,which consist of hundreds of fragments. To address this problem,pulse-field gel electrophoresis (PFGE) has been developed to enable the separation of large DNA fragments. PFGE providesa chromosomal restriction profile typically composed of 5 to20 distinct, well-resolved fragments ranging from ~10–800kilobases (kb). The relative simplicity of the RFLP profilesgenerated by PFGE facilitates application of the procedure inidentification and epidemiological survey of bacterial pathogens. Fingerprinting,which combines PFGE with Southern transfer and hybridization,has been widely used in studying the tuberculosis nosocomialoutbreak in human immunodeficiency virus (HIV)-positive populations(Goering .,2004).

Friday, November 18, 2011

Antibiotics-producing microorganisms

There has reports mentioned that antibiotic production is a feature of several kinds of soil bacteria and fungi and may represent a survival mechanism whereby organisms can eliminate competition and colonize a niche (Jensen et al., 1997; Talaro and Talaro, 1996). Although both fungal and bacterial species are known to produce antibiotics, fungi tend to produce mostly broad-spectrum activities but more antibiotics are produced by bacteria (Salyers and Whitt, 2001).
Oskay et al. (2004) showed that actinomycetes have the capability to synthesize many different biologically active secondary metabolites such as antibiotics, herbicides, pesticides, anti-parasitic, and enzymes like cellulase and xylanase used in waste treatment. Actinomycetes are the most widely distributed groups of microorganisms in nature. They are attractive, bodacious and charming filamentous gram-positive bacteria. They make up in many cases, especially under dry alkaline conditions, a large part of the microbial population of the soil (Athalye et al., 1981; Goodfellow and Williams, 1983; Lacey, 1973 and 1997; Nakayama, 1981; Waksman, 1961). Based on several studies among bacteria, the actinomycetes are noteworthy as antibiotic producers, making three quarters of all known products, the Streptomyces are especially prolific (Lacey, 1973; Lechevalier, 1989; Locci, 1989; Saadoun and Gharaibeh, 2003; Waksman, 1961).
Actinomycetes can be isolated from soil and marine sediments. The soil actinomycetes have been important sources of antibiotics. For example, about 1% of soil actinomycetes produce streptomycin, first discovered in the 1940s, whereas daptomycin producers were discovered only after screening nearly 107 actinomycetes. Most of the antibiotics in use today are derivatives of natural products of actinomycetes and fungi (Butler and Buss, 2006; Newman and Cragg, 2007). Antibiotics produced by actinomycetes have been evolving for ~1 billion years (Baltz, 2005 and 2006), and fitness has been tested by the ability to penetrate other microbes and inhibit the target enzymes, macromolecules or macromolecular structures (Baltz, 2008).
The ability of actinomycetes to make secondary metabolites with different useful properties is widely exploited. Two thirds of the antibiotics produced by microorganisms are made by actinomycetes. In particular, genus of Streptomyces is remarkable in this aspect, representing about 80% of the actinomycete antibiotics (Borodina et al., 2005).
Microbial natural products are the origin of most of the antibiotics. The discovery of penicillin in the 1940s was followed by the discovery of a huge number of antibiotics from microbes, in particular from members of the actinomycetes and fungi. Actinomycetes have traditionally been the most prolific group in antibiotic production. Fungi are another rich source of antibiotics (Peláez, 2006).
Anupama et al. (2007) reported that actinomycetes have been isolated from different soils, plant materials, water and marine sediments (Mincer et al., 2002). At least 90% of the population among actinomycetes isolated from soils have been reported to be Streptomyces spp. Among microorganisms, actinomycetes are the important source for bioactive metabolites especially antibiotics (Bérdy, 2005).

Thursday, November 17, 2011

Plasmid analysis

Plasmids are small, self-replicating circular DNA found in many bacteria.These often encode genes related to antibiotic resistance and certain virulence factors. In epidemiological studies, relatedness of isolated pathogenic bacterial strains can be determined from the number andsize of plasmids the bacteria carry. Plasmid profile analysis was among the earliest nucleic acid-based techniques applied to the diagnosis of infectious diseases and has proven useful in numerous investigations . This method has also been widely utilized for tracking antimicrobial resistance during nosocomial outbreaks. In studies of the epidemiology of plasmids, analysis of restriction fragments has proved valuable. This technique is widely used to monitor the spread of resistance-encoding plasmids between organisms and between hospitals, communities, or even countries.The weakness of the analysis is inherent in the fact that plasmids are mobile, extrachromosomal elements, not part of the chromosomal genotype. Because plasmids can be spontaneously lost from or readily acquired by a host stain, epidemiologically related isolatescan exhibit different plasmid profiles(van et al., 2007).

Wednesday, November 16, 2011

PB19 infection in transplantation

The first report of PVB19 infection after transplantation was published in 1986 [7]. Since then, numerous cases of PVB19 infections after solid-organ transplan-tation (SOT) and hematopoietic stem cell transplantation (HSCT) have been reported. Anemia is the predominant clinical manifestation. However, PVB19 has also been associated with hepatitis, pneumonitis, myocarditis, and allograft dysfunction. Nonetheless, the full spectrum of clinical manifestations of PVB19 infection among transplantat recipients is not well characterized.

The suppression of the RBC population that clinically results in anemia as the hallmark of PVB19 infection is consistent with the cellular tropism of this virus [71]. PVB19 infects erythroid progenitor cells by binding to the receptor known as the P antigen [71]. Subsequent PVB19 replication in erythroid progenitor cells leads to cellular lysis [72], which is characteristically manifested as pure red cell aplasia on bone marrow examination.
Immunocompetent individuals respond to PVB19 infection by producing virus-specific Ig [73–75]. Experimental studies have demonstrated that the generation of PVB19- specific Ig is temporally accompanied by reduction in the degree of parvoviremia [75]. The impairment in immunity that results
from pharmacologic immunosuppression limits the ability of transplant patients to produce neutralizing antibody, which leads to persistent PVB19 infection that manifests as chronicanemia. Not surprisingly, almost all patients in transplants patients series had chronic anemia, many patients did not possess PVB19-specific Ig at the onset of clinical disease, and almost all transplant patients without PVB19 IgM had parvoviremia. Demonstrates that the spectrum of clinical illness related to PVB19 is broad. This reflects the ability of PVB19 to infect other cells [76]. The cardiotropism of PVB19 is suggested by its association with myocarditis [77–79] and left ventricular
dysfunction [80] and by the demonstration of PVB19 DNA in fetal myocardial cells [81]. These data support the suggestion that myocarditis may occur in transplant patients with PVB19 disease, and this may be misdiagnosed as acute rejection and could result in death from cardiogenic shock [17, 28, 43]. The most likely cardiac target of PVB19 is the endothelium [81–83], because endothelial cells in small cardiac vessels also carry P antigen [54]. Likewise, endothelial infection could serve as the mechanism for PVB19-associated thrombotic microangiopathy [54].
Studies of parvoviruses that infect animals demonstrate the virions in various organs [84]. Parvovirus related to Aleutian mink disease was detected in alveolar cells in mink with acute interstitial pneumonitis [85]. Intact Aleutian mink disease parvoviral DNA has also been detected in glomeruli [85]. These animal data support the suggestion that PVB19 is a potential cause of pneumonitis [22, 31, 68], hepatitis [28, 58, 63, 66, 86], and collapsing glomerulopathy [26] in humans. Indeed, PVB19 has been demonstrated in the renal tissue and blood of patients with collapsing glomerulopathy and in hepatocytes of a patient with fibrosing cholestatic hepatitis [58]. Nevertheless, the reported associations between PVB19 and organ-specific syndromes do not definitely indicate causality.
The inability of transplant patients to mount sufficient anti-PVB19 Ig could present a diagnostic dilemma and delay treatment in patients seen at centers who rely on serological examination for the diagnosis PVB19. All except 1 of the patients who did not have PVB19 IgM detected had positive PCR assay results, suggesting the clinical utility of this molecular assay. Our
observation suggests that a negative PVB19 IgM serological test result does not rule out the diagnosis of PVB19 infection, and PCR should be used whenever a diagnosis of acute PVB19 infection is suspected in immunocompromised patients. Among patients who are highly suspected to have PVB19 disease but whose peripheral blood PCR assay result is negative, the diagnosis may be confirmed by bone marrow examination.
If feasible, reduction in immunosuppression should be a part of the treatment of PVB19 disease. Theoretically, this would allow the immune system to mount specific immunity against PBV19. The observation that parvoviremia ceases with generation of Ig [75] led to the current practice of intravenous Ig
treatment of PVB19. Intravenous Ig contains PVB19-specific antibodies. However, the dose and duration of treatment are not standardized. Clinical relapses are commonly observed (i.e. 1 relapse occurs for every 4–5 patients treated), which suggests that the patient experiences a continued state of severe immunosuppression and that there is a need to further reduceimmunosuppression or administer intravenous Ig for a longer period to neutralize parvoviremia. The rarity of this infection limits the conduct of a prospective trial to assess the optimal dose and duration of treatment.
In conclusion, PVB19 can cause rare but significant infectious complication after transplantation. The predominant clinical manifestation of PVB19 disease is anemia, although organ invasive manifestations, such as hepatitis, myocarditis, and pneumonitis, can be observed. However, whether these organ specific syndromes are causally linked to PVB19 infection remains to be proven. A high index of suspicion is advised when patients present with refractory and severe anemia after transplantation. In this clinical setting, PVB19 infection should be considered in the differential diagnosis, together with the other, more likely causes, such as an adverse reaction to treatment,blood loss, and anti-erythropoietin antibody, among others. In this regard, PCR may be a more useful noninvasive test for the confirmation of the diagnosis, because the PVB19 serological test results of many transplant patients are negative at the onset of clinical disease.
A retrospective study of parvovirus B19 antibody titres 2 to 3 years after bone marrow transplantation showed persisting IgG, suggesting that persistence of B19 antibody depends on prior recipient, but not donor, immunity.1
allogeneic peripheral blood stem cell transplantation (PBSCT(consistently results in severe immunodeficiency. It is known that human parvovirus B19 can persist in red blood cell precursors in the bone marrow of immunocompromised patients. An infection can cause severe complications including chronic bone marrow failure or pure red cell aplasia because of the inability of patients to produce neutralizing antibodies against the virus.3 The major route of transmission of parvovirus B19 is inhalation of respiratory droplets from infected people. Both of these patients were treated in special air-filter rooms designed to create a pathogen-reduced environment and no clinical infections were in family members or hospital staff. There is evidence for parvovirus B19 transmission via blood products, PBSC or bone marrow.4, 5 Patients required erythrocyte infusions during conditioning before transplantation. Parvovirus B19 is known to be a frequent contaminant of blood products,6 but as parvovirus B19 screening is not part of the routine control of blood products is not routinely followed in many countriesand so infection via this route cannot be excluded. Another route of infections in those patients is reactivation of old infection. with parvovirus B19 during immunosuppressive conditioning and further immunosuppression. This concept is supported by several reports both in PBSCT and in solid organ transplantation.7
parvovirus B19-induces erythematous infection in immuncompromised patients early after PBSC transplantation and should be considered in the differential diagnosis of acute GvHD of the skin. It highlights the importance of excluding the possibility of a viral infection before initiating treatment for acute GvHD. Furthermore, parvovirus B19 infection should be considered in cases of late anaemia after PBSC or bone marrow transplantation, occurring in patients known to be seropositive for parvovirus B19 IgG.
Lectures on applied clinical microbiology
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