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Biotechologyis the science of utilizing living systems to benefit humankind. In recent years, the ability to directly alter an organism’s genome throughgeneticengineeringhas been made possible due to advances inrecombinant DNA technology,which allows researchers to createrecombinant DNA moleculeswith new combinations...
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Molecular cloninginvolves methods used to construct recombinant DNA and facilitate their replication in host organisms. These methods include the use ofrestriction enzymes(to cut both foreign DNA andplasmid vectors),ligation(to paste fragments of DNA together), and the introduction of recombinant DNA into a host organi...
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Blue-white screeningallows selection of bacterial transformants that contain recombinant plasmids using the phenotype of areporter genethat is disabled by insertion of the DNA fragment.
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Genomic librariescan be made by cloning genomic fragments from one organism into plasmid vectors or into bacteriophage.
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cDNA librariescan be generated to represent the mRNA molecules expressed in a cell at a given point.
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Transfectionof eukaryotic hosts can be achieved through various methods usingelectroporation,gene guns,microinjection,shuttle vectors, andviral vectors.
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Finding a gene of interest within a sample requires the use of a single-strandedDNA probelabeled with a molecular beacon (typically radioactivity or fluorescence) that can hybridize with a complementary single-stranded nucleic acid in the sample.
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Agarose gel electrophoresisallows for the separation of DNA molecules based on size.
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Restriction fragment length polymorphism(RFLP)analysis allows for the visualization by agarose gel electrophoresis of distinct variants of a DNA sequence caused by differences in restriction sites.
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Southern blotanalysis allows researchers to find a particular DNA sequence within a sample whereasnorthern blotanalysis allows researchers to detect a particular mRNA sequence expressed in a sample.
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Microarray technologyis a nucleic acid hybridization technique that allows for the examination of many thousands of genes at once to find differences in genes or gene expression patterns between two samples of genomic DNA or cDNA,
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Polyacrylamide gel electrophoresis (PAGE)allows for the separation of proteins by size, especially if native protein charges are masked through pretreatment with SDS.
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Polymerase chain reactionallows for the rapid amplification of a specific DNA sequence. Variations of PCR can be used to detect mRNA expression (reverse transcriptase PCR) or to quantify a particular sequence in the original sample(real-time PCR).
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Although the development ofSanger DNA sequencingwas revolutionary, advances innext generation sequencingallow for the rapid and inexpensive sequencing of the genomes of many organisms, accelerating the volume of new sequence data.
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The science ofgenomicsallows researchers to study organisms on a holistic level and has many applications of medical relevance.
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Transcriptomicsandproteomicsallow researchers to compare gene expression patterns between different cells and shows great promise in better understanding global responses to various conditions.
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The various –omics technologies complement each other and together provide a more complete picture of an organism’s or microbial community’s (metagenomics) state.
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The analysis required for large data sets produced through genomics, transcriptomics, andproteomicshas led to the emergence ofbioinformatics.
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Reporter genesencoding easily observable characteristics are commonly used to track gene expression patterns of genes of unknown function.
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The use of recombinant DNA technology has revolutionized the pharmaceutical industry, allowing for the rapid production of high-qualityrecombinant DNA pharmaceuticalsused to treat a wide variety of human conditions.
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RNA interferencetechnology has great promise as a method of treating viral infections by silencing the expression of specific genes
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While gene therapy shows great promise for the treatment of genetic diseases, there are also significant risks involved.
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There is considerable federal and local regulation of the development of gene therapies by pharmaceutical companies for use in humans.
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Before gene therapy use can increase dramatically, there are many ethical issues that need to be addressed by the medical and research communities, politicians, and society at large.
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Inanimate items that may harbor microbes and aid in their transmission are calledfomites. The level of cleanliness required for a fomite depends both on the item’s use and the infectious agent with which the item may be contaminated.
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The CDC and the NIH have established fourbiological safety levels (BSLs)for laboratories performing research on infectious agents. Each level is designed to protect laboratory personnel and the community. These BSLs are determined by the agent’s infectivity, ease of transmission, and potential disease severity, as we...
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Disinfectionremoves potential pathogens from a fomite, whereasantisepsisuses antimicrobial chemicals safe enough for tissues; in both cases, microbial load is reduced, but microbes may remain unless the chemical used is strong enough to be asterilant.
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The amount of cleanliness (sterilizationversus high-level disinfection versus general cleanliness) required for items used clinically depends on whether the item will come into contact with sterile tissues (critical item), mucous membranes (semicritical item), or intact skin (noncritical item).
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Medical procedures with a risk for contamination should be carried out in asterile fieldmaintained by properaseptic techniqueto preventsepsis.
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Sterilization is necessary for some medical applications as well as in the food industry, where endospores ofClostridium botulinumare killed throughcommercial sterilizationprotocols.
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Physical or chemical methods to control microbial growth that result in death of the microbe are indicated by the suffixes-cideor-cidal(e.g., as withbactericides,viricides, andfungicides), whereas those that inhibit microbial growth are indicated by the suffixes-stator-static(e.g.,bacteriostatic,fungistatic).
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Microbial death curvesdisplay the logarithmic decline of living microbes exposed to a method of microbial control. The time it takes for a protocol to yield a 1-log (90%) reduction in the microbial population is thedecimal reduction time, orD-value.
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When choosing a microbial control protocol, factors to consider include the length of exposure time, the type of microbe targeted, its susceptibility to the protocol, the intensity of the treatment, the presence of organics that may interfere with the protocol, and the environmental conditions that may alter the effect...
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Heat is a widely used and highly effective method for controlling microbial growth.
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Dry-heat sterilizationprotocols are used commonly in aseptic techniques in the laboratory. However,moist-heat sterilizationis typically the more effective protocol because it penetrates cells better than dry heat does.
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Pasteurizationis used to kill pathogens and reduce the number of microbes that cause food spoilage.High-temperature, short-time pasteurizationis commonly used to pasteurize milk that will be refrigerated;ultra-high temperature pasteurizationcan be used to pasteurize milk for long-term storage without refrigeration.
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Refrigeration slows microbial growth; freezing stops growth, killing some organisms. Laboratory and medical specimens may be frozen on dry ice or at ultra-low temperatures for storage and transport.
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High-pressure processing can be used to kill microbes in food. Hyperbaric oxygen therapy to increase oxygen saturation has also been used to treat certain infections.
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Desiccationhas long been used to preserve foods and is accelerated through the addition of salt or sugar, which decrease water activity in foods.
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Lyophilizationcombines cold exposure and desiccation for the long-term storage of foods and laboratory materials, but microbes remain and can be rehydrated.
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Ionizing radiation, including gamma irradiation, is an effective way to sterilize heat-sensitive and packaged materials.Nonionizing radiation, like ultraviolet light, is unable to penetrate surfaces but is useful for surface sterilization.
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HEPAfiltration is commonly used in hospital ventilation systems and biological safety cabinets in laboratories to prevent transmission of airborne microbes.Membrane filtrationis commonly used to remove bacteria from heat-sensitive solutions.
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Heavy metals, including mercury, silver, copper, and zinc, have long been used for disinfection and preservation, although some have toxicity and environmental risks associated with them.
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Halogens, including chlorine, fluorine, and iodine, are also commonly used for disinfection. Chlorine compounds, includingsodium hypochlorite,chloramines, andchlorine dioxide, are commonly used for water disinfection. Iodine, in bothtinctureandiodophorforms, is an effective antiseptic.
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Alcohols, including ethyl alcohol and isopropyl alcohol, are commonly used antiseptics that act by denaturing proteins and disrupting membranes.
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Phenolicsare stable, long-acting disinfectants that denature proteins and disrupt membranes. They are commonly found in household cleaners, mouthwashes, and hospital disinfectants, and are also used to preserve harvested crops.
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The phenolic compoundtriclosan, found in antibacterial soaps, plastics, and textiles is technically an antibiotic because of its specific mode of action of inhibiting bacterial fatty-acid synthesis..
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Surfactants, including soaps and detergents, lower the surface tension of water to create emulsions that mechanically carry away microbes. Soaps are long-chain fatty acids, whereas detergents are synthetic surfactants.
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Quaternary ammonium compounds(quats) are cationic detergents that disrupt membranes. They are used in household cleaners, skin disinfectants, oral rinses, and mouthwashes.
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Bisbiguanidesdisrupt cell membranes, causing cell contents to gel.Chlorhexidineandalexidineare commonly used for surgical scrubs, for handwashing in clinical settings, and in prescription oral rinses.
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Alkylating agentseffectively sterilize materials at low temperatures but are carcinogenic and may also irritate tissue.Glutaraldehydeando-phthalaldehydeare used as hospital disinfectants but not as antiseptics.Formaldehydeis used for the storage of tissue specimens, as an embalming fluid, and in vaccine preparation to ...
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Peroxygens, includinghydrogen peroxide,peracetic acid,benzoyl peroxide, and ozone gas, are strong oxidizing agents that produce free radicals in cells, damaging their macromolecules. They are environmentally safe and are highly effective disinfectants and antiseptics.
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Pressurized carbon dioxide in the form of asupercritical fluideasily permeates packaged materials and cells, forming carbonic acid and lowering intracellular pH. Supercritical carbon dioxide is nonreactive, nontoxic, nonflammable, and effective at low temperatures for sterilization of medical devices, implants, and tra...
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Chemical preservatives are added to a variety of foods.Sorbic acid,benzoic acid,propionic acid, and their more soluble salts inhibit enzymes or reduce intracellular pH.
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Sulfitesare used in winemaking and food processing to prevent browning of foods.
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Nitritesare used to preserve meats and maintain color, but cooking nitrite-preserved meats may produce carcinogenic nitrosamines.
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Nisinandnatamycinare naturally produced preservatives used in cheeses and meats. Nisin is effective against gram-positive bacteria and natamycin against fungi.
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Chemical disinfectants are grouped by the types of microbes and infectious agents they are effective against.High-level germicideskill vegetative cells, fungi, viruses, and endospores, and can ultimately lead to sterilization.Intermediate-level germicidescannot kill all viruses and are less effective against endospores...
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The effectiveness of a disinfectant is influenced by several factors, including length of exposure, concentration of disinfectant, temperature, and pH.
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Historically, the effectiveness of a chemical disinfectant was compared with that of phenol at killingStaphylococcus aureusandSalmonella entericaserovar Typhi, and aphenol coefficientwas calculated.
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Thedisk-diffusion methodis used to test the effectiveness of a chemical disinfectant against a particular microbe.
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Theuse-dilution testdetermines the effectiveness of a disinfectant on a surface.In-use testscan determine whether disinfectant solutions are being used correctly in clinical settings.
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Antimicrobial drugsproduced by purposeful fermentation and/or contained in plants have been used as traditional medicines in many cultures for millennia.
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The purposeful and systematic search for a chemical “magic bullet” that specifically target infectious microbes was initiated by Paul Ehrlich in the early 20th century.
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The discovery of thenatural antibiotic, penicillin, by Alexander Fleming in 1928 started the modern age of antimicrobial discovery and research.
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Sulfanilamide, the firstsynthetic antimicrobial, was discovered by Gerhard Domagk and colleagues and is a breakdown product of the synthetic dye, prontosil.
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Antimicrobial drugs can bebacteriostaticorbactericidal, and these characteristics are important considerations when selecting the most appropriate drug.
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The use ofnarrow-spectrumantimicrobial drugs is preferred in many cases to avoidsuperinfectionand the development of antimicrobial resistance.
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Broad-spectrumantimicrobial use is warranted for serious systemic infections when there is no time to determine the causative agent, when narrow-spectrum antimicrobials fail, or for the treatment or prevention of infections with multiple types of microbes.
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Thedosageandroute of administrationare important considerations when selecting an antimicrobial to treat and infection. Other considerations include the patient’s age, mass, ability to take oral medications, liver and kidney function, and possible interactions with other drugs the patient may be taking.
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Antibacterial compounds exhibitselective toxicity, largely due to differences between prokaryotic and eukaryotic cell structure.
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Cell wall synthesis inhibitors, including theβ-lactams, theglycopeptides, andbacitracin, interfere with peptidoglycan synthesis, making bacterial cells more prone to osmotic lysis.
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There are a variety of broad-spectrum, bacterial protein synthesis inhibitors that selectively target the prokaryotic 70S ribosome, including those that bind to the 30S subunit (aminoglycosidesandtetracyclines) and others that bind to the 50S subunit (macrolides,lincosamides,chloramphenicol, andoxazolidinones).
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Polymyxinsare lipophilic polypeptide antibiotics that target the lipopolysaccharide component of gram-negative bacteria and ultimately disrupt the integrity of the outer and inner membranes of these bacteria.
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The nucleic acid synthesis inhibitors rifamycins andfluoroquinolonestarget bacterial RNA transcription and DNA replication, respectively.
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Some antibacterial drugs areantimetabolites, acting as competitive inhibitors for bacterial metabolic enzymes.Sulfonamidesandtrimethoprimare antimetabolites that interfere with bacterial folic acid synthesis.Isoniazidis an antimetabolite that interferes with mycolic acid synthesis in mycobacteria.
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Because fungi, protozoans, and helminths are eukaryotic organisms like human cells, it is more challenging to develop antimicrobial drugs that specifically target them. Similarly, it is hard to target viruses because human viruses replicate inside of human cells.
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Antifungal drugsinterfere with ergosterol synthesis, bind to ergosterol to disrupt fungal cell membrane integrity, or target cell wall-specific components or other cellular proteins.
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Antiprotozoan drugsincrease cellular levels of reactive oxygen species, interfere with protozoal DNA replication (nuclear versus kDNA, respectively), and disrupt heme detoxification.
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Antihelminthic drugsdisrupt helminthic and protozoan microtubule formation; block neuronal transmissions; inhibit anaerobic ATP formation and/or oxidative phosphorylation; induce a calcium influx in tapeworms, leading to spasms and paralysis; and interfere with RNA synthesis in schistosomes.
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Antiviral drugsinhibit viral entry, inhibit viral uncoating, inhibit nucleic acid biosynthesis, prevent viral escape from endosomes in host cells, and prevent viral release from infected cells.
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Because it can easily mutate to become drug resistant, HIV is typically treated with a combination of severalantiretroviral drugs, which may includereverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, and drugs that interfere with viral binding and fusion to initiate infection.
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Antimicrobial resistanceis on the rise and is the result of selection of drug-resistant strains in clinical environments, the overuse and misuse of antibacterials, the use of subtherapeutic doses of antibacterial drugs, and poor patient compliance with antibacterial drug therapies.
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Drug resistance genes are often carried on plasmids or in transposons that can undergo vertical transfer easily and between microbes through horizontal gene transfer.
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Common modes of antimicrobial drug resistance include drug modification or inactivation, prevention of cellular uptake or efflux, target modification, target overproduction or enzymatic bypass, and target mimicry.
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Problematic microbial strains showing extensive antimicrobial resistance are emerging; many of these strains can reside as members of the normal microbiota in individuals but also can cause opportunistic infection. The transmission of many of these highly resistant microbial strains often occurs in clinical settings, b...
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TheKirby-Bauer disk diffusiontest helps determine the susceptibility of a microorganism to various antimicrobial drugs. However, thezones of inhibitionmeasured must be correlated to known standards to determine susceptibility and resistance, and do not provide information on bactericidal versus bacteriostatic activity,...
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Antibiograms are useful for monitoring local trends in antimicrobial resistance/susceptibility and for directing appropriate selection of empiric antibacterial therapy.
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There are several laboratory methods available for determining theminimum inhibitory concentration (MIC)of an antimicrobial drug against a specific microbe. Theminimal bactericidal concentration (MBC)can also be determined, typically as a follow-up experiment to MIC determination using the tube dilution method.
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Current research into the development of antimicrobial drugs involves the use of high-throughput screening and combinatorial chemistry technologies.
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New technologies are being developed to discover novel antibiotics from soil microorganisms that cannot be cultured by standard laboratory methods.
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Additional strategies include searching for antibiotics from sources other than soil, identifying new antibacterial targets, using combinatorial chemistry to develop novel drugs, developing drugs that inhibit resistance mechanisms, and developing drugs that target virulence factors and hold infections in check.
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In aninfection, a microorganism enters a host and begins to multiply. Some infections causedisease, which is any deviation from the normal function or structure of the host.
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Signsof a disease are objective and are measured.Symptomsof a disease are subjective and are reported by the patient.
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Diseases can either benoninfectious(due to genetics and environment) orinfectious(due to pathogens). Some infectious diseases arecommunicable(transmissible between individuals) orcontagious(easily transmissible between individuals); others arenoncommunicable, but may be contracted via contact with environmental reservo...
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Nosocomial diseasesare contracted in hospital settings, whereasiatrogenic diseaseare the direct result of a medical procedure
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Anacute diseaseis short in duration, whereas achronic diseaselasts for months or years.Latent diseaseslast for years, but are distinguished from chronic diseases by the lack of active replication during extended dormant periods.
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The periods of disease include theincubation period, theprodromal period, theperiod of illness, theperiod of decline, and theperiod of convalescence. These periods are marked by changes in the number of infectious agents and the severity of signs and symptoms.
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Koch’s postulatesare used to determine whether a particular microorganism is a pathogen.Molecular Koch’s postulatesare used to determine what genes contribute to a pathogen’s ability to cause disease.
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Virulence, the degree to which a pathogen can cause disease, can be quantified by calculating either theID50orLD50of a pathogen on a given population.
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