Introduction to Microbiology for Nurses PDF

Introduction to Microbiology NURS 114 Dr. S. F. Gyasi 1  Course Outline This course is designed to give student knowledge about microbial organisms, how infections and infestation are transmitted and how to disinfect and sterilize materials. The cou...

Introduction to Microbiology NURS 114 Dr. S. F. Gyasi 1  Course Outline This course is designed to give student knowledge about microbial organisms, how infections and infestation are transmitted and how to disinfect and sterilize materials. The course has a concurrent practical component to enable students view micro-organisms using the light microscope. The course will offer student basic knowledge on the role, types, sources and control of disease- causing micro-organisms. 2  Unit I: History of microbiology; ecology and habitat, Definitions and terminologies Microbiology, Pathology, Immunology, Infection, endemic, pandemic, sporadic, epidemiology, Susceptibility, Sensitivity, Specificity, Inoculation, Immunisation etc) 3  Unit II: Classification of microorganisms; principal groups of microbes; protozoa, bacteria. Rickettsiae, viruses, fungi, 4  Unit II Cont’d Chlamydia, spirochetes and their characteristic, Disease transmission cycle, Trilogic relationship and how to break it, defence mechanism 5  Unit III: Conditions suitable for growth of microbes- aerobes and anaerobes; pathogens Identification of microbes: microscopy; simple staining, gram staining, ziehl-neelsen staining, 6 Unit IV: Interpretation of common laboratory results, Infection prevention (primary, secondary, tertiary), nosocomial asepsis (medical and surgical), universal precautions: hand washing, use of protective clothing, disposal of waste materials, contamination, high level disinfection and sterilization 7 Unit V: People at risk of infections (providers, clients/patients, visitors, significant others, communities, environment) and risk factors (direct contact with blood and other body fluids, biological hazards, bacteria, fungi viruses, parasites 8 STUDENT EVALUATION 40% = Continuous Assessment (Attendance, Assignments, Quizzes, Mid Semester Examination) 60% = End of Semester Examination 9  Reading List Bell, B. (2016), Immunology made ridiculously simple. MedMaster Inc; Betsy, Tom & Keogh, Jim (2005).Microbiology demystified. New York: McGraw-Hill, Brooks, G.F., Butel, J.S., & Morse, S. A., (2004). Jawetz, Melnic & Adelbergs Medical Micrbiology, McGraw Hill. 10  Chen, E. M. & Sanjay S. Katsuri (2010), Deja Review Microbiology & Immunology, (2nd ed) , New York, McGraw-Hill Gladwin, M & Trattler, B (2007).Clinical Microbiology made ridiculously simple. (4th ed). Miami: Med Master, Inc. Hawley, L. Clarke B & Ziegler R. J (2010) Microbiology and Immunology (Board Review Series) , : LWW Levinson, W. (2014)., Review of Medical Microbiology and Immunology, (11thed) New York McGraw-Hill 11  Murray, P. R & Rosenthal, K. S. (2005). Medical microbiology. (5th ed). Philadelphia: Elsevier Mosby. Nester, E. W. (2007). Microbiology: a human perspective. (5th ed). Boston: McGraw-Hill. Prescott, L. M.; Harley, J. P., & Klein, D. A.,(2005) : Microbiology. (6th ed). McGraw- Hill. Boston Tortora, G. J & Funke, B. R. (2006). Microbiology: an introduction. (9th ed). San Francisco: Pearson Education, Inc. 12  What is a Microbiology? It is the study of organisms too small to be seen with the naked eye Smaller than 0.1mm Includes bugs, germs, viruses, protozoan, bacteria, animalcules etc Why study Microbiology as a Nurse? 14  Nomenclature Carolus Linnaeus (1735) Genus species By custom once mentioned can be abbreviated with initial of genus followed by specific epithet. E. coli When two organisms share a common genus, they are related. What organisms considered to be microbial cells and studied in microbiology 1. BACTERIA/ARCHAEA 2. FUNGI 3. ALGAE 4. PROTOZOA 5. Viruses(although not a cellular entity but an intracellular pathogen) 6. Prions (a biochemical anomaly—misfolded proteins) 7. Helminths Worms (multicellular) The History of Microbiology 17 Spontaneous Generation  Spontaneous Generation – From earliest time, people had believed in spontaneous generation existed among microbes – i.e., Living organisms could develop from non living matter – Even the great Aristotle (384-322 BC) believed some of the simpler vertebrate could arise by spontaneous generation  Supported by: – Aristotle (384-322 BC) – Believed that simple invertebrates could arise by spontaneous generation – John Needham (1713- 1781) – – Boiled mutton broth, then sealed and still observed growth after a period of time Lazarro Spallanzani (1729-1799) Improved on Needham’s experimental design by first sealing glass flaks that contained water and seed;  If the sealed flask was placed in boiling water for 3-4 hrs, no growth occurred. He proposed that, air carried germs to the culture medium But also commented that external air may be required for the growth of animals already in the medium 22 The Microscopy Era The first person to describe microorganism accurately was by the amateur microscopist, Anton van Leeuwenhoek (1632 - 1723) His discovery renewed the controversy Several investigators attempted to counter this arguments. Schwann, and his colleagues (1830s) – Air allowed to a enter flask but only after passing through a heated tube or sterile wool – The flask remained sterile-No growth – John Tyndall (1820-1893) – Prevented dust in in flask of media but did not heat  no growth. Despite all these experiment to disprove Spontaneous generation, Felix Pouchet claimed in (1859) conclusively proving that microbial growth could occur without air contamination  Disproved by: This was settled finally by an Italian Physician Francesco Redi and Louis Pasteur It was Redi who carried out a series of experiments on decaying meat and its ability to produce maggot spontaneously.  Louis Pasteur (1822 - 1895) First filtered air into cotton and found out that objects resembling plants spores had been trapped If a piece of the cotton was placed in a sterile medium after air had been filtered through it, microbial growth appeared Next, he placed nutrient solution in a flask, heated their necks in flames and drew them out in a variety of curves whiles keeping their ends open to the atmosphere.  He pointed out that, no growth occurred because dust and germs had been trapped on the walls of the curved neck. If the necks were broken, growth commenced immediately. Pasteur had not only resolved the controversy by 1861 but also had shown how to keep a solution sterile  Role of Micoorganisms in Disease Franscesco Stelluti Believed that disease was caused by invisible forces known as Miasma He observed bees and weevils using a microscope in the early 1600s  Recognition of Microbial Role in Disease Although Francastoro and a few others had suggested that invisible organisms produced diseases, most believed diseases was due to causes such as supernatural and poisonous vapours called miasmas And imbalances between the 4 humours Blood Phlegm Yellow bile(Choler) Black bile (Melancholy) Lead to diseases had been widely accepted since the time of the Greek physician Galen (129-199)  Support of the germ theory of disease began to accumulate in the early 19th Century.  Demonstrations that microorganisms cause disease Agostino Bassi (1773 - 1856) – First showed that a microorganism can cause a disease when he demonstrated that silkworm disease was caused by a fungus – He also suggested that, many diseases were caused by microbial infections M. J. Berkeley (1845) – Proved that, the Great Potato Blight of Ireland was caused by a Fungus  Demonstrating microbial cause disease Louis Pasteur – Following his success with the study of fermentation, Pasteur was asked by the French government to investigate that the pébrine disease of silkworms that was affected the silk industry – After several years of research, he showed that the disease was caused by a protozoan parasite – The disease was controlled by raising caterpillar from eggs produced by healthy moths  Joseph Lister (1827 - 1912) – Indirect evidence that microorganisms caused disease came from the work of the English surgeon Joseph Lister on the prevention of wound infection – He developed a system of surgery designed to prevent microorganisms from entering wounds – phenol sprayed in air around surgical incision – Decreased number of post-operative infections in patients – his published findings (1867) transformed the practice of surgery 37  Charles Chamberland (1851 - 1908) – Identified viruses as disease-causing agents – Tobacco Mosaic Virus Edward Jenner (1798) – Used a vaccination procedure to protect individuals from smallpox Louis Pasteur – Developed other vaccines including those for chicken cholera, anthrax, and rabies  Terminologies Definitions and terminologies Pathology, Immunology, Infection, epidemic, endemic, pandemic, sporadic, epidemiology, Susceptibility, Sensitivity, Specificity, Inoculation, Immunisation etc) 39 What is pathology Infection, endemic, pandemic, sporadic, epidemiology, Susceptibility, Sensitivity, Specificity, Inoculation, Immunisation 40  What is Pathology Pathology is the study of the links between diseases and the basic science Pathologist is a person identifying diseases, based on the examination of cells and tissues. removed from the body 41  What is a Disease? A disease is a physical or functional disorder of normal body systems that places an individual at increased risk of adverse consequences Diseases are diagnosed by physicians or other health care providers through a combination of tools When a disease is diagnosed, treatment is given to prevent or ameliorate complications and to improve prognosis 42  Immunology Is the branch of biomedical/bioloical science concerned with the response of the organism to antigenic challenge. It is also the recognition of self from non-self, and all of the biological (in vivo), serological (in vitro), and physical chemical aspects of immune phenomena defense mechanisms They include all physical, chemical and biological properties of the organism which reduce it's susceptibility to foreign organisms, material, etc. 43  What is an infection An infection is a process where by a microorganism (bacteria, fungi, protozoan etc) enters an individual host, attach and multiply and do some damage. Sometimes, the infection itself may not produce the damage but the damage is illicited by our immune responese 44  Outbreaks, Epidemics, and Pandemics — What’s the difference? Outbreaks, epidemics, and pandemics all involved in infectious disease, but there are key differences to consider when epidemiologists classify disease cases. 45  Outbreaks The occurrence of a disease is classified as an outbreak when it occurs in greater numbers than normally expected. It could occur in a larger area or region, a smaller community, or even a specific location, such as a hospital. It can last from days to years or occur seasonally year-after-year. 46  It is generally thought that all cases of an outbreak are related in some way and in general, are more localized than epidemics. Eg: Cholera in Ghana killing 100s of people, Cryptosporidium-1993 in Milwaukee caused 403,000 people to be ill and 100 deaths; Pertussis-2010 in California caused 9,477 people to be ill; 10 infant deaths 47  Epidemics An epidemic is an increase in the number of disease cases expected in an area, but is generally a more widespread situation than an outbreak. Though size of the area or region or numbers of disease are not the only factors when classifying an outbreak or epidemic, an epidemic generally has more disease cases over a larger region than an outbreak. The severe acute respiratory syndrome (SARS Cov1) epidemic in 2003 affected over 8,000 people worldwide and took the lives of 774 people. 48  Pandemics A pandemic is an epidemic that occurs over large areas such as entire continents and affects a large proportion of the population. Pandemics have the potential to spread to multiple countries, especially in today’s ease of global transportation. Eg: COVID 19, 49  What is a sporadic Disease Medical Definition of sporadic. 1 : occurring occasionally, singly, or in scattered instances sporadic diseases — compare endemic, epidemic sense. 2 : arising or occurring randomly with no known cause sporadic Buruli ulcer or Cholera disease 50  What is Epidemiology What is Epidemiology? Epidemiology is the study of the determinants, distribution and frequency of disease (who gets the disease and why) The study of the distribution and determinants of health-related states or events in specified populations, and the application of this study to control of health problems. 51  Epidemiologists study sick people They also study healthy people to determine the crucial difference between those who get the disease and those who are spared. The Epidemiologists study exposed people and non-exposed people to determine the crucial effect of the exposure 52  What is Susceptibility susceptible.... Well, with susceptible meaning "likely to be influenced or affected by" that is probably going to be the case. If you're susceptible to flattery, and someone wants something from you, all they have to do is give you a compliment or two and you'll do what they want. 53  susceptible. If you are susceptible to something such as infections it means you are likely to become sick with these things.... Well, with susceptible meaning "likely to be influenced or affected by" that is probably going to be the case. 54 Sensitivity and Specificity 55 What is Inoculation and Immunisation 56  Bacteria Prokaryotes Peptidoglycan cell walls Binary fission For energy, they use organic chemicals, inorganic chemicals, or photosynthesis Figure 1.1a BACTERIAL CELL Prokaryotes 1. NO nucleus 2. NO membrane bound organelles (just ribosomes) 3. ALL are unicellular 4. Smaller than eukaryotic cells 5. Forerunner to eukaryotic cells (smaller and more simple) 6. DNA – single strand and circular 7. Ex: ALL Bacteria  Prokaryotic Morphology (Shapes) BACTERIA are extremely minute, chlorophyll-free, unicellular bodies, which multiply by simple binary fission. They have also been designated at various times by such names as " germs, microbes, micro-organisms ". Of these, the term " bacterium " has been the one most widely applied, and it is perhaps unfortunate that it has also been adopted as a generic name for a particular group of rod-shaped forms. The bacteria are placed at the lower limit of the scale of living bodies and they have affinities with the lower forms of both plant- life, e.g., the algae, moulds and fungi, and animal life, e.g., the protozoa.  The size of bacteria The size of bacteria is measured in micrometer (m) or micron () (1 micron or micrometer is one thousandth of a millimeter) and varies from 0.1  to 16-18 . Most pathogenic bacteria measure from 0.1 to 10 . The other units of measurement of microorganisms are millimicron (m) or nanometer (nm) (one millionth of a millimeter) and 1 Angstrom (Å) (one tenth of nanometer).  Shape of Bacteria. The variations in the appearance of the different bacteria were recognized by the earliest observers. Leeuwenhoek (1683) described clearly bacillary and spiral forms, and attempts were later made to classify the bacteria on morphological appearances. While these classifications are now of little value, it is interesting to note that many of the terms employed in these early schemes are still in use. The main forms now recognized are the coccus, bacillus, vibrio, spirillum, actinomyces and spirochaete  Bacterial Morphology Bacteria only take a few basic shapes, which are found in many different groups. Bacterial cells don’t have internal cytoskeletons, so their shapes can’t be very elaborate. Shape: coccus (spheres) and bacillus (rods). Spirillum (spiral) is less common. Aggregation of cells: single cells, pairs (diplo), chains (strepto), clusters (staphylo). Thus we have types such as diplococcus (pair of spheres) and streptobacillus (chain of rods).  A Coccus is a spherical form ; in some cases, such as the pneumococcus, one diameter is definitely longer than the other, so that the form is oval rather than round. There are other characteristic arrangements, such as long chains, pairs or small clusters which are produced mainly by the mode of division, are found.  A Bacillus is a cylindrical rod ; Great variation is found among the different members in the proportion of length to transverse diameter. Division always takes place by transverse fission after previous elongation and constriction at the site of the division. The form is variable and may be a straight rod with square ends, or the long axis may be bent and the ends rounded or pointed. The size of the different bacilli varies considerably ; some are very large while others approximate to the coccal forms and are sometimes referred tô as "cocco-bacilli". The term Bacillus has now been adopted as the generic name for a group of Gram-positive, sporing, aerobic, rod-shaped organisms, While the term Bacterium is used as the generic name of a group of Gram-negative rod-shaped forms.  This is perhaps unfortunate, as there must consequently arise a certain amount of confusion from the use of these two terms, each of which has both a specific and a general application. When microbial names are used in generic sense these names are given a capital and are printed in italics, Eg Mycobaterium ulcerans or Bacillus athracis  A Vibrio is a cylindrical form slightly bent or curved on itself ; A single cell may frequently present the shape of a comma. A Spirillum is a form closely related to the vibrio and is characterized by a number of bends or spirals along the long axis. An Actinomyces is a form differing from the above in that filaments and branching are found. These forms are sometimes referred to as the higher bacteria. A Spirochaete is an elongated, flexible organism twisted spirally around its long axis and exhibiting motility without possessing flagella. It resembles in some respects both the bacteria and the protozoa.  Morphology of Bacteria Bacteria are intracellular free-living organisms having both DNA and RNA. Their biological properties and predominant reproduction by binary fission relates them to prokaryotes. Spherical (cocci) Rod-shaped (bacteria, bacilli, and clostridia) Spiral-shaped (vibriones, spirilla, spirochaetes) Spherical (cocci) bacteria 1. Micrococci 2. Diplococci 3. Streptococci 4. Staphylococci 5. Tetracocci 6. Sarcinae Representatives of pathogenic cocci 1 2 1.Scanning Electron Micrograph of Streptococcus pneumoniae 2.Scanning electron micrograph of a Staphylococcus aureus Electron Micrograph of Neisseria gonorrhoeae  More Rod-shaped bacteria Some Bacteria include those microorganisms, as a rule, do not produce spores (E.coli, Salmonella, Shigella). But some others including Bacilli anthracis, Clostridia tetani, and Clostridia botulinum) include organisms the majority of which produce spores. Size of rod-shaped bacteria varies 2-10 μm: small rods are 2- 4 μm; long rods are 5-10 μm. 1 2 3 ARRANGEMENT OF ROD-SHAPED BACTERIA  Rod-shaped bacteria 2 1 1. Single Rod and 2 Streptobacillus  SPIRAL FORMS 1. Vibrios – are cells, which resemble a comma in appearance (curved rods). Typical representative of this group is Vibrio cholerae. 2. Spirilla – are coiled forms of bacteria. Pathogenic species: (i) Spirillum minus – which is responsible for a disease in humans transmitted through the bite of rats – rat-bite fever – sodoku; (ii) Helicobacter pylori – causative agent of ulcer disease of stomach. 2 1  SPIROCHAETES Treponema – exhibits, thin, flexible cells with 6-14 regular twists. The size of Treponema varies from 10-18 μ (T. pallidum). Leptospira – are characterized by very thin cell structure. The leptospirae form 12-18 regular coils (primary spirals) (L. interrogans) and C- or S- shape according secondary twist. Borrelia – have large irregular spirals, the number of which varies from 3 to 10. (B. recurrentis and B.persica).  3. Spirochaetes – are flexuous spiral forms which include: (1) Treponema (T. pallidum), (2) Borrelia (B.recurrentis) (3) Leptospira (L. interrogans) 1 3 2 3  Fungi Eukaryotes Chitin cell walls Use organic chemicals for energy Moulds and mushrooms are multicellular consisting of masses of mycelia, which are composed of filaments called hyphae Yeasts are unicellular Figure 1.1b  Protozoa Eukaryotes Absorb or ingest organic chemicals May be motile via pseudopods, cilia, or flagella Most free some parasites Figure 1.1c  Viruses Acellular Consist of DNA or RNA core Core is surrounded by a protein coat Coat may be enclosed in a lipid envelope Viruses are replicated only when they are in a living host cell Figure 1.1e  What is a Viruses? Viruses are microorganisms-hence they cannot be seen with the unaided eye. A virus is an infectious agent made up of nucleic acid (DNA or RNA) wrapped in a protein coat called a capsid. Viruses have no definite nucleus, no organelles, no cytoplasm or cell membrane. Viruses have either DNA or RNA but NOT both What is a virus? ‘Infectious particle’ Genetic material Protein coat  Historical Perspectives of Viruses The existence of viruses was first suspected in the 19th century when it was shown that filtered extract of infective material passed through filters small enough to stop all known bacteria could still be infectious, Hence the ‘virus’ (Latin for poisonous liquid) concept was first introduced. However, viral diseases such as smallpox and poliomyelitis had been known to affect mankind for many centuries. Subsequent to the discovery of viruses, the next major step in elucidating their role in human disease was the invention of the electron microscope, followed by cell culture and now molecular diagnostic techniques to detect the presence of viruses in infected material.  In 1983 acquired immunodeficiency syndrome (AIDS) was discovered and the study of virology came to the lime light…. Millions of dollars have been spent by pharmaceutical companies in discovering drugs to treat AIDS; a by-product has been that our understanding of virus replication and pathogenesis has improved substantially. This has resulted in new antiviral drugs becoming available to treat other viral infections. The availability of rapid and sensitive molecular diagnostic techniques and effective antiviral drug therapy means that patients can now be treated in real time.  Types of Viruses Viral cells have 2 main types of genetic information – DNA and RNA Viruses may contain either DNA or RNA A DNA virus injects its DNA into the host cell, and the DNA can control the cell directly – Examples – HPV; Herpes; Epstein-Barr A virus with RNA is called a retrovirus – retroviruses inject their RNA into a host cell and force the cell to make new viral DNA, which may then be integrated into the cell’s normal DNA – Examples – HIV, rhinovirus (common cold, SARS CoV 2 and flu)  Viral Taxonomy i. Viruses have either an RNA or DNA genome (never both) and are classified in families on the basis of their genome (RNA or DNA) and whether it is single or double stranded (SS or DS). ii. Single-stranded RNA viruses are further split on the basis of whether they carry a negative (-ve RNA) or a positive (+ve RNA) strand as this affects their replication strategy. iii. As a rule of thumb all DNA viruses except those belonging to Parvoviridae are double stranded and all RNA viruses except those belonging to Reoviridae are single stranded iii. Other features taken into consideration are their size and shape, and the presence or absence of a lipid envelope, which some viruses acquire as they bud out of cells. iv. RNA viruses generally tend to be enveloped and have outer proteins (required for attachment to the cell surface) projecting out of this lipid envelope, e.g. haemagglutinin (HA) of influenza A virus. -The viral genome is packaged within a nucleoprotein (capsid) which consists of a repetition of structurally similar amino acid sub-units. vii. The viral genome and the capsid are together referred to as nucleocapsid. The viral nucleoprotein or capsid gives the virus its shape (helical or icosahedral).  Properties of a virus A virus is a very small, infectious, obligate intracellular parasite. Virus particles cannot survive outside its host Viruses by themselves cannot reproduce A susceptible and permissive cellular host is needed for viruses to reproduce All viruses must make mRNA that can be translated by host ribosomes Structure of Virus Protein Capsule – Surrounds genetic material DNA RNA No means of independent metabolism or growth No means of independent http://library.thinkquest.org/C0123260/basic%20kno wledge/DNA.htm reproduction – Dependent on host life form – Can evolve or change over time http://www.laportecounty.org/departments/ani mal_shelter/rabie_virus.html Basic virus structure DNA OR RNA Basic virus structure DNA OR + RNA Basic virus structure DNA OR + Capsid Protein RNA Basic virus structure DNA OR + Capsid Protein RNA Basic virus structure DNA OR + Capsid Protein Nucleocapsid RNA Basic virus structure DNA Capsid OR RNA + Protein Nucleocapsid Basic virus structure DNA Capsid OR + Protein Nucleocapsid = RNA Basic virus structure DNA Capsid OR + Protein Nucleocapsid = RNA + Basic virus structure DNA Capsid OR + Protein Nucleocapsid = RNA Lipid membrane + Glycoproteins Basic virus structure DNA Capsid OR + Protein Nucleocapsid = RNA Lipid membrane + Enveloped virus Glycoproteins Capsid symmetry Icosahedral Helical  Viral Replication Viruses are obligate intracellular pathogens and require cellular enzymes to help them replicate. Unlike bacteria, which replicate by binary fission, viruses have to ‘disassemble’ their structure before they can replicate. The steps of viral replication can be broadly divided into: (i) Attachment, (ii) Cell entry, (iii) Virus disassembly or uncoating, Transcription and Translation of viral genome, (vi)viral Assembly (Maturation) (vii) Release. The steps of viral replication (i) Attachment, (ii) Cell entry, (iii) Virus disassembly or uncoating, Transcription and Translation of viral genome, (iv)Viral Assembly and (v) Release. The steps of viral replication  Attachment The first step in the replication cycle is the attachment of the virus particle to the cell surface. To do this specific viruses use specific cellular receptors on the cell surface and therefore are very specific in the cell type that they can infect – this gives them the ‘cell tropism’ and is important in disease pathogenesis (i.e. why some viruses affect certain organs only). Influenza viruses use the haemagglutinin (HA) protein to attach to the sialic acid-containing oligosaccharides on the cell surface.  Sometimes viruses may use more than one cell receptor, for example HIV uses the CD4 receptor to attach to the CD4 T-helper cells, but it also uses a chemokine receptor CCR5 as a co- receptor. SARS CoV 2 uses ACE 2-Angionstine Co-Enzyme 2 It is now believed that most viruses use more than one receptor on the cell surface in a sequential binding process  Cell entry Viruses may enter the cell directly by endocytosis or, for enveloped viruses, by fusion of their lipid envelope with the cell membrane. Virus disassembly or uncoating Before the virus can replicate, the viral genome has to be exposed by removal of the associated viral proteins. This is usually mediated by the endocytosis viral particle merging with cellular lysosomes; The resulting drop in pH dissociates the viral genome from its binding protein.  Transcription and Translation of viral genome How a virus replicates is dictated by the structure of its viral genome. (a) Viruses containing SS+RNA use their +RNA as mRNA and utilize the cell’s ribosomes and enzymes to translate the information contained in this +RNA to produce viral proteins. One of the first proteins to be produced is a RNA-dependent RNA polymerase, which then transcribes viral RNA into further RNA genomes. These viruses, because they can subvert the cellular system for their own replication, do not need to carry the information for the initial replication enzymes within their genome. (b) Viruses containing SS-RNA need to convert first to a +RNA strand, which is then used as a mRNA template for translation or direct transcription to the genomic -RNA. They therefore need to carry a viral-specific RNA- dependent RNA polymerase. (c) DS RNA viruses have to first convert the -RNA strand of the DS RNA into a complementary +RNA to be used as mRNA. The +RNA strand of the DS RNA acts as a template for viral genome replication. These viruses also need to carry the RNA-dependent RNA polymerase to initiate the first steps of viral replication  Retroviruses are unique SS+RNA viruses. Instead of using the SS+RNA as an mRNA template, the RNA is first transcribed into complementary DNA by an RNA-dependent DNA polymerase in a process called reverse transcription (hence the name, retro reverse). The normal transcription is always from DNA to RNA. Further transcription then occurs as for other SS DNA viruses. DNA virus mRNA is transcribed from the DS DNA viruses in a similar fashion to cellular DNA replication. These viruses can therefore completely depend upon the cellular process to replicate. The genome of these viruses (e.g. cytomegalovirus (CMV), Epstein–Barr virus (EBV) needs to carry information to code for the virus specific proteins only.  Regulatory proteins and those required for viral DNA synthesis are coded early on and the later proteins are generally structural proteins. Single stranded DNA viruses are first converted into double stranded, and then mRNA is transcribed as for the DS DNA viruses.  Viral Assembly and Release Before the virus particle can be released its proteins and genome have to be assembled within the cell as a ‘viral package’. This process may require the cell to alter viral proteins by glycosylation. Viral release may occur either through cell death or through viral budding from the cell membrane. Enveloped viruses use the “budding from the cell membrane” mechanism to acquire their lipid envelope at this stage. Viral enzymes such as the neuraminidase (NA) of influenza viruses (which acts on the sialic-acid bond on the cell surface to release the infectious virus particle) and may be required for the viruses released via budding  Viral pathogenesis Viral pathogenesis can be described as the process by which the virus interacts with its host to produce disease. As this is a process which involves virus–host interaction, both viral and host factors have a bearing on the pathogenesis of viral disease.  Virus as Pathogen – disease causing agent Mumps Chicken Pox Measles / AIDS, Rabies, Cold, Hanta,Hepatitis, Flu, Polio, SARS,Hemorrhagic Small Pox Fever, (Ebola) Yellow Fever,SARS CoV2 etc  Algae Eukaryotes Cellulose cell walls Use photosynthesis for energy (primary producers) Produce molecular oxygen and organic compounds Metabolically diverse Examples of algae acting as a mammalian pathogen are known Eg: Protothecosis. Figure 1.1d  Protothecosis is a disease found in dogs, cats, cattle, and humans caused by a type of green alga known as Prototheca that lacks chlorophyll. 118  Multicellular Animal Parasites Eukaryote Multicellular animals Parasitic flatworms and round worms are called helminths. Microscopic stages in life cycles. Figure fluke Microbial Nutrition & Growth 120  Growth in Batch Culture Typically, a batch culture passes through four distinct stages: – Lag stage – Logarithmic (exponential) growth – Stationary stage – Death stage Growth in Batch Culture  Mean Generation Time and Growth Rate The mean generation time (doubling time) is the amount of time required for the concentration of cells to double during the log stage. It is expressed in units of minutes. 1 Growth rate (min-1) = mean generation time Mean generation time can be determined directly from a log plot of bacterial concentration vs time after inoculation Measurement of Microbial Growth Turbidity – Based on the diffraction or “scattering” of light by bacteria in a broth culture – Light scattering is measured as optical absorbance in a spectrophotometer – Optical absorbance is directly proportional to the concentration of bacteria in the suspension  Growth in Continuous Culture A “continuous culture” is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate. This method is accomplished in a device called a chemostat. Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained. Factors that Influence Microbial Growth Growth vs. Tolerance – “Growth” is generally used to refer to the acquisition of biomass leading to cell division, or reproduction – Many microbes can survive under conditions in which they cannot grow – The suffix “-phile” is often used to describe conditions permitting growth, whereas the term “tolerant” describes conditions in which the organisms survive, but don’t necessarily grow  For example, a “thermophilic bacterium” grows under conditions of elevated temperature, while a “thermotolerant bacterium” survives elevated temperature, but grows at a lower temperature Factors that Influence Microbial Growth Obligate (strict) vs. Facultative – “Obligate” (or “strict”) means that a given condition is required for growth – “Facultative” means that the organism can grow under the condition, but doesn’t require it – The term “facultative” is often applied to sub- optimal conditions – For example, an obligate thermophile requires elevated temperatures for growth, while a facultative thermophile may grow in either elevated temperatures or lower temperatures  Factors that Influence Microbial Growth Temperature – Most bacteria grow throughout a range of approximately 20 ºC, with the maximum growth rate at a certain “optimum temperature” – Psychrophiles: Grows well at 0ºC; optimally between 0ºC – 15ºC – Psychrotrophs: Can grow at 0 – 10ºC; optimum between 20 – 30ºC and maximum around 35ºC – Mesophiles: Optimum around 20 – 45ºC – Moderate thermophiles: Optimum around 55 – 65 ºC – Extreme thermophiles (Hyperthermophiles): Optimum around 80 – 113 ºC Factors that Influence Microbial Growth pH – Acidophiles: Grow optimally between ~pH 0 and 5.5 – Neutrophiles Grow optimally between pH 5.5 and 8 – Alkalophiles Grow optimally between pH 8 – 11.5 Factors that Influence Microbial Growth Salt concentration – Halophiles require elevated salt concentrations to grow; often require 0.2 M ionic strength or greater and some may grow at 1 M or greater; example, Halobacterium – Osmotolerant (halotolerant) organisms grow over a wide range of salt concentrations or ionic strengths; for example, Staphylococcus aureus Factors that Influence Microbial Growth Oxygen concentration – Strict aerobes: Require oxygen for growth (~20%) – Strict anaerobes: Grow in the absence of oxygen; cannot grow in the presence of oxygen – Facultative anaerobes: Grow best in the presence of oxygen, but are able to grow (at reduced rates) or in the absence of oxygen – Aerotolerant anaerobes: Can grow equally well in the presence or absence of oxygen – Microaerophiles: Require reduced concentrations of oxygen (~2 – 10%) for growth  Nutrient Requirements Carbon source – Autotroph Can use CO2 as a sole carbon source (Carbon fixation) – Heterotroph Requires an organic carbon source; cannot use CO2 as a carbon source  Nutrient Requirements Nitrogen source – Organic nitrogen Primarily from the catabolism of amino acids – Oxidized forms of inorganic nitrogen Nitrate (NO32-) and nitrite (NO2-) – Reduced inorganic nitrogen Ammonium (NH4+) – Dissolved nitrogen gas (N2) (Nitrogen fixation)  Nutrient Requirements Phosphate source – Organic phosphate – Inorganic phosphate (H2PO4- and HPO42-)  Nutrient Requirements Sulfur source – Organic sulfur – Oxidized inorganic sulfur Sulfate (SO42-) – Reduced inorganic sulfur Sulfide (S2- or H2S) – Elemental sulfur (So)  Nutrient Requirements Special requirements – Amino acids – Nucleotide bases – Enzymatic cofactors or “vitamins”  Microbiological Media Chemically defined vs. Complex media – Chemically defined media The exact chemical composition is known e.g. minimal media used in bacterial genetics experiments – Complex media Exact chemical composition is not known Often consist of plant or animal extracts, such as soybean meal, milk protein, etc. Include most routine laboratory media, e.g., tryptic soy broth  Microbiological Media Selective media – Contain agents that inhibit the growth of certain bacteria while permitting the growth of others – Frequently used to isolate specific organisms from a large population of contaminants Differential media – Contain indicators that react differently with different organisms (for example, producing colonies with different colours) – Used in identifying specific organisms  What is Koch’s Postulates It is the causal relationship between microorganisms and the diseases they cause Koch injected healthy mice with materials from diseased (anthrax) animals and the mice became ill. After transferring anthrax from inoculation into a series of 20 mice, he inoculated a piece of spleen containing the anthrax bacillus in beef serum. 142  What is Koch’s Postulates The bacilli grew, reproduced and produced spores When the isolated bacillus or spores were injected into the mice, anthrax developed. 143  His criteria for proving the causal relationship between microorganism and the specific disease they cause are known as the Koch’s Postulate; 144  Koch’s Postulates Microorganism must be present in every case of the disease but absent in healthy organism. The suspected microorganism must be isolated and grown in a pure culture The same disease must result when the isolated microorganism is inoculated in a health host The same microorganism must be isolated again from the host host  Application of the Koch’s Postulates The linkages between Mycobacterium ulcerans and Buruli Ulcer Disease What is Buruli Ulcer It is caused by What are the risk factors 146  (A) (B) Source-WHO, 2008 (C) (D) Assessing the susceptibility of arsenic exposed ICR mice to the development of Buruli Ulcer Isolation of MU from human tissue  Animal Model  Experimental animal (80 - 6 wks old healthy ICR Mice) A-E Mice put into 8 grps of 10 (n=10) Labelled-A to H Mortality and emaciation (7 days)  Water containing arsenic preparation Arsenic trichloride - Levels in accordance to BU endemic communities  A-E- (0.8, 1.6, 2.4, 3.6 or 4.8 mg/L) F- [No Arsenic-(Ordinary tape water) No MU)] control 1 or Proper control G-(Only Arsenic) Control 2 H-(Only MU) Control 3  Observation for 14 days  M. ulcerans Isolation and culture for inoculum preparation  Tissue collection and transport Diagnosing M. ulcerans in tissue by PCR/Microscopy Picking of swab PCR/Microscopy to confirm M. ulcerans  Culture of MU (L-J media) Bacterial sub culture(L-J media) o Microscopy (AFB) o PCR  Inoculum preparation PCR/Microscopy on Inoculum Staining (AFB) Slantz of L-J Media Preparation of MU Inoculum and Injection of mice after 14 days (Arsenic)  MU inoculum prepared using PBS at pH 7  Inoculum adjusted to 5 MacFarland standard  Intraplantarly injection of mice (A-E & Control 3) with 0.05mls of Inoculum = 7.5 x 106 CFU  0.05 mls of inoculum was also cultured  Observation of both experimental and control mice until day 98 (+14)=122 days  Results……..  Clinical Evaluation After day 112 Inflammation and erythema after 58 days of MU exposure (72 days of As exposure)  Clinical Evaluation After day 112 Inflammation and erythema after 58 days of Lesions with ulcerated genitals of mice after MU exposure (72 days of As exposure) 98 days of MU exposure (112 days of As Exposure) Oedema with ulcerated surface (112 days)  Clinical Evaluation After day 112 Lesions with ulcerated genitals of mice after Inflammation and erythema after 58 days of 98 days of MU exposure (112 days of As MU exposure (72 days of As exposure) Exposure) A previously positive lesion that has Oedema with ulcerated surface (112 days) amputated (Day 112)  Haematological Analysis after 112 days  3 mice each from each group sacrificed  About 1ml of blood taken into EDTA tube for analysis  WBC, RBC ,HGB, HCT, MCV, MCH, MCHC, RDW-CV & RWD- SD,PLT, MPV, PDW  Histopathological Analysis after 112 days  Liver, spleen, lungs, heart and kidney harvested from the 3 mice in (10% formalin)  Histopathological Evaluation 1. Tissue grossing 2. Tissue processing 2. Tissue waxing 6. Examination of slide 5. Drying of slide after 4. Microtomy Photomicrograph of the liver (A) of arsenic exposed MU inoculated ICR Mice after 98 days (112 days of arsenic) Normal liver (control) (A) Experimental Mice (MU and As) Photomicrograph of the liver (A) & spleen (B) of arsenic exposed MU inoculated ICR Mice after 98 days (112 days of arsenic) Normal liver (control) (A) Experimental Mice (MU and As) Normal spleen (control) (B)Experimental Mice (MU a Photomicrograph of the liver of MU only inoculated ICR Mice after 112 days prior to MU positive lessions Normal liver Experimental Mice (Only MU inoculated) Photomicrograph of the liver of MU only inoculated ICR Mice after 112 days prior to MU positive lessions Experimental Mice (Only MU Normal liver inoculated) Normal spleen Experimental spleen (MU only in  BU in MU only after day 122 MU positive reported in mice After 122 days(50 days before the onset of MU positive lesions in As + MU exposure groups) Histopathological & haematological assessment similar to day 112  Most E. coli strains are harmless, but a few pathogenic (disease-causing) strains exist, causing food poisoning. A common source is ground meat, but it gets on unwashed vegetables as well. Related enteric bacteria: Salmonella, Shigella. Cause food poisoning. Chickens carry Salmonella in their guts instead of E. coli. 164  Lenses and Bending of Light When light travels from one medium to another, bending occurs- Refraction Refraction index is the measure of how greatly a substance slows the velocity of light passing through it. The direction and magnitude of the bend is determined by the refractive indexes of the 2 media forming the interface 165  When light passes from air into a glass, and returns to air 166  A prism is able to bend light rays because it has a different refractive index compared to air and that the light strikes it at an angel Lenses are made up of a collection of prims 167 Compound light microscopy Basic parts – Eyepieces (ocular lens) – Base – Condenser – Iris diaphragm – Objective lens – Body tube – Mechanical stage – Adjustment knobs Microbial Staining Techniques Preparation and staining of Specimen Although living organisms can directly examined with light microscope, They often must be stained i. To increase visibility ii. Accentuate specific morphological features iii. Preserve specimen for future use  Fixation The stained cells seen in the microscope should resemble living cells as closely as possible FIXATION is therefore the process by which the internal and external features of a cell and microorganisms are preserved and fixed in position Significance of fixation It inactivate enzymes that might disrupt cell morphology And toughen the cell structure so they do not change during staining and observation  Types of Fixation i. Heat fix- By gently flaming bacteria smears on a slide or by air dry in a hot oven This preserve the overall morphology but not structure Chemical fix- By chemical fixatives This penetrates the cell and react with cellular component usually proteins and lipids to render them inactive, insoluble and immobile Stains All dyes are salts – Ionize Cationic Anionic Techniques – Single dyes – Multiple dyes Chemical Makeup of Stains Benzene = organic compound Chromophore = color Auxochrome = ionization properties Benzene + Chromophore = Chromogen – Chromogen is a colored compound only Auxochrome with Chromogen allows the dye to form salt compounds that adhere to cells. Examples Acid Red Methylene Blue Giemsa  Basic Dyes Cationic in nature Work best in basic pH Bind to –vely charged molecules like nucleic acid and many proteins Because bacteria cell wall are –vely charged, basic dyes are used to stain Egs – Methylene Blue – Crystal Violet – Carbol Fuchsin – Safranin – Malachite Green  Acidic Dyes Anionic in nature Work best in acidic pH Bind to +vely charged cell wall Egs: – Picric Acid – Nigrosin – India Ink – Eosin  Staining Methods/Types Negative Stain Simple Stain Differential Stains – Group Gram Stain Acid Fast Stain – Special Structures Capsule Stain Endospore Stain Flagella Stain  Slide Preparation Clean slide LABEL !!! Smear in circle – Broth – Solid + H20 Air dry first Heat fix (usually) – Kill organism – Adhere to slide – Accepts dye Problems – Too thick – Wash off specimen  1. Negative Stain Acid Dye (-) chromogen Repelled by (-) cell wall Cells – Colorless – Seen against dark background  2. Simple Stain One reagent used Soak smear 30-60 seconds Rinse with H20 Background stained Bacteria stained  3. Simple Stain Basic dye  (+) chromogen  (-) cell wall  Shows morphology Size Shape Arrangement  Acid Fast Bacillus Test Another differential staining method Because do not stain easily, a harsher means are applied Heating with a mixture of basic fusion and Phenol known as Ziehl-Neelsen method  3. Differential Stains Two or more reagents Distinguish – Bacterial groups – Specific Structures Eg: – Gram stain – Acid Fast Stain  Gram stain Theory The Theory: The Gram Stain works, because bacteria can be divided into two main groups based on the morphology of their cell wall. Bacterial cells are either gram-positive (purple) or gram-negative (red) 187 Gram Stain General Theory  Time Frame 1) 1 minute 2) 1 minute 3) 15 seconds 4) 1 minute Rinse with water between each step  Principles of Communicable Diseases Epidemiology  Definition of Epidemiology Epidemiology is the study of the distribution and determinants of health-related states and events in populations, and the application of this study to control health problems (Last, 1983). Epidemiologic triad Demographic characteristics Biological characteristics Socioeconomic characteristics Host Agent Environment Biological agents Physical agents Physical environment Chemical agents Biological environment Nutrient agents Social environment Mechanical agents Social agents Infectious Disease Model Host Pathogen disease Environment  Definition of communicable diseases A communicable disease is an illness due to a specific infectious (biological) agent or its toxic products capable of being directly or indirectly transmitted from man to man, from animal to man, from animal to animal, or from the environment (through air, water, food, etc..) to man.  Importance of Studying Communicable Diseases Epidemiology Changes of the pattern of infectious diseases Discovery of new infections The possibility that some chronic diseases have an infective origin.  Terminology and Definitions Exotic Infection Sporadic Contamination Attack rate Infestation Primary/secondary cases Contagious disease Zoonosis, epizootic and Incidence and prevalence of enzootic infectious diseases Nosocomial infection Epidemic Opportunistic infection Endemic Eradication Hyperendemic Elimination holoendemic Pandemic Terminology and Definitions (cont.) Virulence Incubation period Reproductive rate of Infectivity period infection Serial interval Host Latent period Vector (source) Transmission Probability Reservoir ratio Infection Infection is the entry and development or multiplication of an infectious agent in the body of man or animals. An infection does not always cause illness. There are several levels of infection (Gradients of infection): – Colonization (S. aureus in skin and normal nasopharynx) – Subclinical or inapparent infection (polio) – Latent infection (virus of herpes simplex) – Manifest or clinical infection contamination The presence of an infectious agent on a body surface, on or in clothes, beddings, toys, surgical instruments or dressings, or other articles or substances including water and food Infestation It is the lodgment, development and reproduction of arthropods on the surface of the body or in the clothing, e.g. lice, itch mite. This term could be also used to describe the invasion of the gut by parasitic worms, e.g. ascariasis. Contagious disease A contagious disease is the one that is transmitted through contact. Examples include scabies, trachoma, STD and leprosy. Host A person or an animal that affords subsistence or lodgement to an infectious agent under natural conditions. Types include: an obligate host, definitive (primary) host, intermediate host and a transport host. Vector of infection An insect or any living carrier that transports an infectious agent from an infected individual or its wastes to a susceptible individual or its food or immediate surroundings. Both biological and mechanical transmissions are encountered. Reservoir Any person, animal, arthropod, plant, soil, or substance, or a combination of these, in which an infectious agent normally lives and multiplies, on which it depends primarily for survival, and where it reproduces itself in such a manner that it can be transmitted to a susceptible host. It is the natural habitat of the infectious agent. Incidence and prevalence of infectious diseases Incidence of an infectious disease: number of new cases in a given time period expressed as percent infected per year (cumulative incidence) or number per person time of observation (incidence density). Prevalence of an infectious disease: number of cases at a given time expressed as a percent at a given time. Prevalence is a product of incidence x duration of disease, and is of little interest if an infectious disease is of short duration (i.e. measles), but may be of interest if an infectious disease is of long duration (i.e. chronic hepatitis B). Epidemic “The unusual occurrence in a community of disease, specific health related behavior, or other health related events clearly in excess of expected occurrence” (epi= upon; demos= people) Epidemics can occur upon endemic states too. Endemic It refers to the constant presence of a disease or infectious agent within a given geographic area or population group. It is the usual or expected frequency of disease within a population. (En = in; demos = people)  Hyperendemic and holoendemic The term “hyperendemic” expresses that the disease is constantly present at high incidence and/or prevalence rate and affects all age groups equally. The term “holoendemic” expresses a high level of infection beginning early in life and affecting most of the children population, leading to a state of equilibrium such that the adult population shows evidence of the disease much less commonly than do the children (e.g. malaria) Pandemic and Exotic An epidemic usually affecting a large proportion of the population, occuring over a wide geographic area such as a section of a nation, the entire nation, a continent or the world, e.g. Influenza pandemics. Exotic diseases are those which are imported into a country in which they do not otherwise occur, as for example, rabies in the UK. Sporadic The word sporadic means “scattered about”. The cases occur irregularly, haphazardly from time to time, and generally infrequently. The cases are few and separated widely in time and place that they show no or little connection with each other, nor a recognizable common source of infection e.g. polio, meningococcal meningitis, tetanus…. However, a sporadic disease could be the starting point of an epidemic when the conditions are favorable for its spread. Attack rates and primary/secondary cases Attack rate: Proportion of non-immune exposed individuals who become clinically ill. Primary (index)/secondary cases: The person who comes into and infects a population is the primary case. Those who subsequently contract the infection are secondary cases. Further spread is described as "waves" or "generations". Zoonosis, epizootic and enzootic Zoonosis is an infection that is transmissible under natural conditions from vertebrate animals to man, e.g. rabies, plague, bovine tuberculosis….. An epizotic is an outbreak (epidemic) of disease in an animal population, e.g. rift valley fever. An Enzotic is an endemic occurring in animals, e.g. bovine TB. Nosocomial infections Nosocomial (hospital acquired) infection is an infection originating in a patient while in a hospital or another health care facility. It has to be a new disorder unrelated to the patient’s primary condition. Examples include infection of surgical wounds, hepatitis B and urinary tract infetions. Opportunistic infection This is infection by organisms that take the opportunity provided by a defect in host defense (e.g. immunity) to infect the host and thus cause disease. For example, opportunistic infections are very common in AIDS. Organisms include Herpes simplex, cytomegalovirus, M. tuberculosis…. Eradication and Elimination Eradication is the termination of all transmission of infection by the extermination of the infectious agent through surveillance and containment. Eradication is an absolute process, an “all or none” phenomenon, restricted to termination of infection from the whole world. The term elimination is sometimes used to describe eradication of a disease from a large geographic region. Disease which are amenable to elimination in the meantime are polio, measles and diphtheria. Reproductive rate of infection: Reproductive Rate of infection (Rr): Potential for an infectious disease to spread. Influential factors include the probability of transmission between an infected and a susceptible individual; frequency of population contact; duration of infection; virulence of the organism and population immune proportion. Dynamics of disease Transmission (Chain of Infection) I II III Source or Reservoir Modes of transmission Susceptible host  (I): Source or Reservoir The starting point for the occurrence of a communicable disease is the existence of a reservoir or source of infection. The source of infection is defined as “the person, animal, object or substance from which an infectious agent passes or is disseminated to the host (immediate source). The reservoir is “any person, animal, arthropod, plant, soil, or substance, or a combination of these, in which an infectious agent normally lives and multiplies, on which it depends primarily for survival, and where it reproduces itself in such a manner that it can be transmitted to a susceptible host. It is the natural habitat of the infectious agent.” Types of reservoirs Reservoir Human Animal Non-living reservoir reservoir reservoir Human reservoir Human reservoir Type: Incubatory cases Primary case Convalescent carriers Index case healthy Secondary cases Duration: According to spectrum of disease: Portal of exit: Temporary Clinical cases Urinary Chronic (mild/severe-typical/atypical) Intestinal Sub-clinical cases Respiratory Latent infection cases others Cases A case is defined as “a person in the population or study group identified as having the particular disease, health disorder, or condition under investigation”  Carriers It occurs either due to inadequate treatment or immune response, the disease agent is not completely eliminated, leading to a carrier state. It is “an infected person or animal that harbors a specific infectious agent in the absence of discernible (visible) clinical disease and serves as a potential source of infection to others. Three elements have to occur to form a carrier state: 1. The presence in the body of the disease agent. 2. The absence of recognizable symptoms and signs of disease. 3. The shedding of disease agent in the discharge or excretions.  Animal reservoirs Zoonosis is an infection that is transmissible under natural conditions from vertebrate animals to man, e.g. rabies, plague, bovine tuberculosis….. There are over a 100 zoonotic diseases that can be conveyed from animal to man.  Reservoir in non-living things Soil and inanimate matter can also act as reservoir of infection. For example, soil may harbor agents that causes tetanus, anthrax and coccidiodomycosis.  (II): Modes of transmission Mode of transmission Indirect Direct transmission transmission Vehicle-borne Direct contact Vector-borne: Droplet infection Mechanical propagative biological Cyclo-prop. Contact with soil Air-borne Cyclo-develop. Inoculation into skin or mucosa Fomite-born Trans-placental (vertical) Unclean hands and fingers (III): Susceptible host An infectious agent seeks a susceptible host aiming “successful parasitism”. Four stages are required for successful parasitism: 1. Portal of entry 2. Site of election inside the body 3. Portal of exit 4. Survival in external environment Virulence and Case Fatality Rate Virulence: is the degree of pathogenicity; the disease evoking power of a micro-organism in a given host. Numerically expressed as the ratio of the number of cases of overt infection to the total number infected, as determined by immunoassay. When death is the only criterion of severity, this is the case fatality rate. Case fatality rate for infectious diseases: is the proportion of infected individuals who die of the infection. This is a function of the severity of the infection and is heavily influenced by how many mild cases are not diagnosed. Serial interval and Infectious period Serial interval: (the gap in time between the onset of the primary and the secondary cases) the interval between receipt of infection and maximal infectivity of the host (also called generation time). Infectious (communicable) period: length of time a person can transmit disease (sheds the infectious agent). Incubation and Latent periods Incubation period: time from exposure to development of disease. In other words, the time interval between invasion by an infectious agent and the appearance of the first sign or symptom of the disease in question. Latent period: the period between exposure and the onset of infectiousness (this may be shorter or longer than the incubation period). Transmission Probability Ratio (TPR) TPR is a measure of the risk of transmission from an infected individual to a susceptible person during a contact. TPR of differing types of contacts, infectious agents, infection routes and strains can be calculated. There are 4 types of transmission probabilities. TPR (cont.) Transmission probabilities: p00: tp from unvaccinated infective to unvaccinated susceptible p01: tp from vaccinated infective to unvaccinated susceptible p10: tp from unvaccinated infective to vaccinated susceptible p11: tp from vaccinated infective to vaccinated susceptible TPR (cont.) To estimate the effect of a vaccine in reducing susceptibility, compare the ratio of p10 to p00. To estimate the effect of a vaccine in reducing infectiousness, compare the ratio of p01 to p00. To estimate the combined effect of a vaccine, compare the ratio of p11 to p00.  Microbial Pathogen and their Control Microorganism are organisms too small to be seen clearly with the unaided eye These are affected by physical and chemical disinfectant  Microbial Control Sterilization: It comes from a latin meaning unable to produce offspring/barren Is a process by which all living cells, visible spores, viruses or viroid are either destroyed or are removed from an object or a habitat. A sterile object is totally free from all viable organism, spores and other infective agents Treatment that kills and remove all living cells Disinfectant: It reduces the total number of microbes on an object or surface but does not completely remove or kill all microbes It is the killing, inhibition or the removal of microorganism that may cause disease Disinfecting agents are agents usually chemical used to carry out disinfection and are only used on inanimate objects. A disinfecting agent usually does not sterilize an object because viable spores and a few objects may remain 235 Sanitation: Reduces the microbial population to levels that is considered safe by public health standards Sanitation is usually used to clean cooking utensils Sanitize: Mechanical process (scrubbing, rinsing etc) that reduces the microbial load on a surface. Sanitizer are the chemical agents employed for this task Eg Soap, hand sanitizers, detergent, etc 236  Anti septic: (Anti=against, Septic=Petrifaction) This is mainly the prevention of infection or sepsis Mild disinfectant, agent, suitable for the skin Normally, a suffix is used to describe the type of antimicrobial agent Cidal: A suffix meaning “the agent kill” Eg. Bacteriocidal, virucidal, parasitocidal, etc  Static: a suffix meaning “the agent that inhibit growth” Eg Fungistatic, bacteriostatic, virustatic etc Pasteurization: This is heating at a temperature below boiling point (60 OC ) suitable to kill spoilage organisms. 238  Physical Methods of Microbial control Moist heat: Exposure to heat (dry and hot) for about 10mins is enough to destroy vegetative cells Unfortunately, the temperature of boiling water 100 deg Cel is not enough to destroy bacteria endospore Therefore boiling water can be used for disinfection of drinking water and objects not harmed by water, but boiling water does not sterilize Thermal death (TD): it is the lowest temperature at which a microbial suspension is killed within 1omins of exposure  Because such a temperature is logarithmic, it is practically not possible to kill all microrganim in a given sample even with extended time, Decithermal reduction time or D value is used. The D value is the time required to kill 90% of microorganisms in a given sample at a specific temperature 240  Dry heat: Here the items to sterilized are placed in a hot oven at 160 to 170 Deg. Cel for 2 to 3 hrs Microbial death results from oxidation of cell Constituents and denaturation of proteins. Although dry heat is less effective than moist heat Clostridium botulinium spores are killed in 5mins at 121Deg. Cel. by moist heat but only after 2 hrs at 160 Deg with dry heat, it has some definite advantages. i. Dry heat does not corrode glass ware and metal plates ii. It can be used to sterilize powders, oils 241  Disadvantages include i Not suitable for heat sensitive materials like many plastics and rubber Low temperature (Freezing) Filtration Radiation Desiccation 242 Chemical agents of Microbial control Qualities of Ideal chemicals include Must be able to kill all microbes within a short time Must not be harmful to the body, cloths, metals or other surfaces Must be effective on all surfaces Must be effective in the presence of proteins and oil The potency must not be affected by changes in pH and temperature  Antimicrobial Chemical Agents All of these denature the protein portions of the microorganisms concerned Phenolics Alcohols Detergents Halogens Heavy metals Aldehydes Sterilizing gases etc