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1 CHAPTER 6 TRANSMISSION OF INFECTION, THE COMPROMISED HOST, EPIDEMIOLOGY, AND DIAGNOSING INFECTIONS WHY IS THIS IMPORTANT? Understanding the ways in which infectious diseases are transmitted and the role of a compromised host in the process, and rapid and correct diagnosis of infections is vital for developing methods to prevent and monitor the spread of disease In order to understand the transmission of disease, it is important to look at: Where pathogens responsible for infection are found Mechanisms of transmission from the environment to a host Mechanisms of transmission from a host to the wider population WHY IS THIS IMPORTANT? The compromised host must be viewed as an integral part of the infection process The more compromised the host, the greater is the risk of successful infection An understanding of the basic principles of epidemiology is critical in preventing the spread of disease Detecting infections is crucial using traditional and modern diagnostics. Genetic engineering and some areas of biotechnology provide tools to diagnose sooner and more reliably 1
2 OVERVIEW Transmission of Infection, the Compromised Host, Epidemiology, and Diagnosing Infections TRANSMISSION THE COMPROMISED EPIDEMIOLOGY DIAGNOSING OF INFECTION HOST INFECTIONS TRANSMISSION OF INFECTION The transmission (spread) of infection is the final requirement for a successful pathogen Two factors affect the spread of infection: Reservoirs of infectious organisms places where pathogens can grow and accumulate Mechanisms of transmission the various ways in which pathogens move from place to place PATHOGEN RESERVOIRS There are three potential reservoirs of pathogens: Humans Sick people easy to identify when symptoms are visible, but difficult when symptoms have yet to develop or have ceased Carriers of infections individuals who will never show symptoms but are still infectious. Other animals Nonliving reservoirs 2
3 ANIMAL RESERVOIRS Diseases transferred from animals to humans are called zoonotic diseases Zoonotic diseases are usually transmitted through direct contact with humans They can also be transmitted through indirect contact such as the waste material of a litter box, fur, feathers, or infected meats Zoonotic diseases can also be indirectly transmitted by vectors ANIMAL RESERVOIRS NONLIVING RESERVOIRS Nonliving reservoirs of infection include water, food, and soil Water is the most dangerous. We cannot live without water Infections of this kind are often seen in countries with poor sanitation and where opportunities for personal hygiene can be limited, leading to high levels of fecal contamination in the water and infections spreading via the fecal-oral route 3
4 MECHANISMS OF TRANSMISSION There are three mechanisms of pathogen transmission: Contact transmission Vehicle transmission Vector transmission CONTACT TRANSMISSION A healthy person is exposed to pathogens by either touching or being in proximity to an infected person or object There are three types of contact transmission: Direct contact transmission Indirect contact transmission Droplet transmission DIRECT CONTACT TRANSMISSION There is no intermediary between infected and uninfected individuals It encompasses such things as touching, kissing, and sexual interactions Pathogens transmitted through direct contact include: Hepatitis A Smallpox Staphylococcus species Herpes virus HIV 4
5 INDIRECT CONTACT TRANSMISSION Takes place through intermediates that are usually nonliving articles: Tissues Handkerchiefs Towels Bedding Contaminated needles (easily transfers HIV and Hepatitis B) Nonliving intermediates that act as the agents of transmission by indirect contact are referred to as fomites DROPLET TRANSMISSION Droplet transmission is seen in the transfer of respiratory diseases such as influenza and whooping cough It can occur through sneezing, coughing, and even laughing Although confined to short distances, the size of the droplet is important Large droplets will fall to the ground quickly, but smaller droplets can stay airborne for long periods The smaller the droplet, the more dangerous it is as an agent of disease VEHICLE TRANSMISSION Vehicle transmission involves pathogens riding along on supposedly clean components Examples of vehicles include: Air Food Water Blood and bodily fluids Drugs and intravenous fluids Air is a difficult vehicle to control Dust uses air as a vehicle and can contain huge numbers of pathogens Microbial spores can also use air to travel from host to host 5
6 VECTOR TRANSMISSION Pathogens are transmitted by carriers, usually arthropods: Fleas Ticks Flies Lice Mosquitoes VECTOR TRANSMISSION There are two types of vector transmission: Mechanical vector transmission pathogens are on vector s body parts and are passively brushed off and onto the host Biological transmission pathogens are within the vector and transmission to the host is through a bite FACTORS AFFECTING DISEASE TRANSMISSION Aside from the overall health, for all people, the disease process is affected by: Age disease levels tend to increase as we age Gender some diseases are more prevalent in one gender or another (e.g. urinary tract infections are seen more in women) Lifestyle poor nutrition can decrease immunocompetence in the host Occupation more infections are seen in health care workers Emotional state a vulnerable emotional state can decrease immunocompetence in the host Climate there is a greater incidence of respiratory infections in colder climates 6
7 PORTALS OF EXIT Many portals of exit are identical to the portals of entry Pathogens use these to exit from a host In exiting, this is usually achieved through bodily secretions such as saliva, sputum, and respiratory droplets However, pathogens can also leave the body via blood, vaginal secretions, semen, urine, and feces PORTALS OF EXIT THE COMPROMISED HOST Spread of disease and pathogens can be dependent on the host The host s ability to mount a defense is referred to as its immunocompetence If host defenses are in some way compromised the potential for damaging infectious disease increases This point is dramatically illustrated in acquired immune deficiency syndrome (AIDS) Other situations which can weaken the immune defenses of the host include lifestyle, occupation, trauma, travel, and aging 7
8 THE COMPROMISED HOST Several groups of people are considered to be vulnerable to infection and therefore immunocompromised to varying degree People with AIDS or genetic immunodeficiency diseases People undergoing chemotherapy or taking broad-spectrum antibiotics Surgical, transplant, and burn patients Premature and newborn infants The elderly Patients on artificial ventilators, with catheters or cannulas NEUTROPENIA Neutropenia is defined as lower-than-normal numbers of neutrophils in the blood: Neutrophils are a form of primary phagocytic defense and an important component of a host s innate immune response The most common cause of profound neutropenia is the administration cytotoxic chemotherapy, cancer or other diseases that damage bone marrow, congenital disorders characterized by poor bone marrow function, viral infections that disrupt bone marrow function, autoimmune disorders that destroy neutrophils or bone marrow cells, and overwhelming infections that use up neutrophils faster than they can be produced NEUTROPENIA The types of infections seen with neutropenia are primarily bacterial and fungal Bacterial infections can begin as soon as neutrophil levels drop Fungal infections are usually only seen after neutrophil levels have been low for a prolonged period of time These infections are often associated with surgical procedures and catheterization 8
9 ORGAN TRANSPLANTATION The immune system recognizes the differences between host organs and transplanted organs Rejection of transplanted organs is suppressed through the administration of drugs These drugs reduce rejection but cause increased susceptibility to infection Infections in organ-transplant patients require treatment with broad spectrum antibiotics Unfortunately, the overuse of these drugs can increase antibiotic resistance and lead to superinfections BURN PATIENTS Burn victims are at risk because of the loss of large areas of the primary physical barrier to infection the skin When skin is lost, there is a greater chance of infection and septicemia Pseudomonas aeruginosa infections are a particular OPPORTUNISTIC INFECTIONS Many infections are caused by opportunistic pathogens These pathogens can be part of the normal flora in our bodies Pathogens can be safe in the specific areas where they normally reside but can move to other parts of the body and cause infection The best example of this type of infection is urinary tract infections (UTIs) Some opportunistic infections can be due to the loss of normal flora in our bodies This type of infection can be due to overuse/improper use of antibiotics with other conditions 9
10 NOSOCOMIAL INFECTIONS Any infection acquired in the hospital or medical facility is called a nosocomial infection Nosocomial infections can affect patients and health care workers There are approximately two million nosocomial infections in the United States each year resulting in approximately 90,000 deaths (twice the number of people killed in traffic accidents) and costs of over $5 billion NOSOCOMIAL INFECTIONS These types of infections are usually associated with intravenous applications such as the following: Catheterization Invasive tests and surgery The same factors used when considering any other type of infection apply to hospital-borne infections. Hospitals must consider: The source of the infection The mode of transmission of the pathogen The susceptibility of the patient to infection Prevention and control NOSOCOMIAL INFECTIONS The most common sites of nosocomial infections are the following: Urinary tract Respiratory tract Surgical wounds The most common pathogens which cause nosocomial infections are: Escherichia coli Enterococcus species Staphylococcus aureus Clostridium difficile Pseudomonas aeruginosa Many of these organisms are resistant to antibiotics including methicillinresistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) 10
11 NOSOCOMIAL INFECTIONS The most common sources of nosocomial infections within the hospital environs are: Other patients Hospital staff Visitors Unsanitary conditions Water supplies Respiratory equipment Catheters PREVENTING NOSOCOMIAL INFECTIONS NOSOCOMIAL INFECTIONS: Prevention and Control Every hospital in the US must have programs in place to address the following concerns: Surveillance of nosocomial infections in patients and staff On-site microbiology laboratory plus standardized isolation procedures Standardized procedures for the use of catheters and hospital equipment Proper decontamination and sanitary procedures Mandatory nosocomial-disease education programs In some cases, infection-control specialist on staff 11
12 EPIDEMIOLOGY Epidemiology is the study of the factors and mechanisms involved in the frequency and spread of disease and other health-related problems Epidemiology can be used not only as a tool to study disease but also as a way to design methods for the control and prevention of diseases INCIDENCE AND PREVALENCE The incidence of a disease is the number of new cases in a set population over a specific period Knowing the incidence level gives information on the spread of a disease The prevalence of a disease is the total number of people infected within a specific population at any given time: Prevalence data measure how seriously and how long a population is affected by a disease MORBIDITY AND MORTALITY RATES Morbidity rate the number of individuals affected during a set period divided by the total population number Mortality rate the number of deaths due to a specific disease divided by the total population number 12
13 MORBIDITY AND MORTALITY RATES Epidemiological studies classify diseases as: Sporadic occurring in random manner; no threat to public health Endemic diseases that are constantly in the population in low numbers Epidemic incidence of a disease suddenly higher than expected Morbidity and mortality rates may increase This may become a more widespread public health problem. Pandemic worldwide epidemic TWO TYPES OF EPIDEMIC Common source epidemic: Arises from contact with contaminated substances It affects a large numbers of people It subsides quickly when the contamination is dealt with Propagated epidemic: It is amplified by person to person contact It remains in the population for a long time It is more difficult to deal with than a common source outbreak HERD IMMUNITY Herd immunity is an important concept in limiting the spread of infection It is conferred to people through vaccination or if they are naturally exposed to the infection and prevents re-infection by the same pathogen When a majority of a population (herd) is immune to an infection there are very few potential hosts and the disease essentially disappears 13
14 HERD IMMUNITY Herd immunity for poliomyelitis is high The vaccine is routinely administered to children so there are few targets available for infection Herd immunity for smallpox is low Smallpox had been eradicated No one is vaccinated for this infection anymore except for the military Since vaccinations have ceased, the number of people immune to smallpox is low and there are many potential targets available for infection HERD IMMUNITY DESCRIPTIVE EPIDEMIOLOGICAL STUDY Concerned with the physical aspects of patients and spread of disease Allows for tracing the outbreak and identifying the first case A descriptive study includes: Data on a number of cases Data on which segment of the population is affected Data on the location of the infection Data on the age, race, marital status, and occupation of the infected population 14
15 ANALYTICAL EPIDEMIOLOGICAL STUDY Focuses on establishing the cause-and-effect relationship Always uses a control group Can be retrospective or prospective Considers factors that occur as the epidemic proceeds TYPES OF EPIDEMIOLOGICAL STUDY Health departments of local and state governments require reports of certain diseases This information has been able to show how the effects on infectious diseases have changed over the years Some diseases are referred to as nationally notifiable meaning that in the United States, they must also be reported to the Centers for Disease Control, the clearing house for epidemiological studies DIAGNOSING INFECTIONS Infectious disease control, prevention, and therapy are highly dependent on quick and correct diagnosis of the species or even strain of pathogen involved There is a basic framework to diagnostic steps which are impacted on by availability of time and resources Key criteria of any diagnostic method are specificity and sensitivity High specificity: identifying a pathogen reliably and correctly High sensitivity: detection of very small amounts of the pathogen or antibodies against it Take samples near the site of infection or with the highest pathogen load and circumvent the patient s normal flora Common samples are blood, urine, feces, sputum, swabs, and biopsy samples Choose appropriate transport media and conditions and label correctly 15
16 GROWTH-BASED DIAGNOSTICS Microorganisms grow differently on different solid or liquid media under aerobic or anaerobic conditions Selective media suppress the growth of some organisms Elective media support the growth of only the organism of interest Differential media show differences between organisms in mixed samples After the mixture of organisms from the sample has been grown the suspected pathogen might be isolated in pure culture Morphology and sometimes the pathogen s physiology is analyzed SEROLOGY-BASED DIAGNOSTICS Identify the pathogen based on certain of its antigens and the antibodies against it Usually blood is used as the sample directly Either isolated or manufactured antibodies are used to detect pathogen antigens or host antibodies in the sample Large amounts of specific, well-characterized antibodies are needed as the target for identification or as a tool in reliable routine diagnostics This demand has been met for some time by advances in biotechnology and the pharmaceutical industry BIOTECHNOLOGY AND HEALTH Biotechnology affects every aspect from food to reproduction to politics Tools of biotechnology are used in everything from manufacturing soaps, to genetically engineering crops, and to potentially turning bacteria into weapons 16
17 BIOTECHNOLOGY AND HEALTH Despite the diversity of cell types in nature most cells have the same basic properties These shared basic characteristics provide opportunities for external manipulation Basic techniques used are genetic engineering, the use of recombinant DNA, and the various specialties that fall under the umbrella name molecular biology HISTORICAL ASPECTS 1953 Watson and Crick define the structure of DNA 1966 Definition of the human genetic code 1970s Recombinant DNA experimentation 1980s Successful recombination of genetic material First production of human drug by bacteria insulin First recombinant vaccine for humans hepatitis B 1990s Human Genome Project started Animals successfully cloned Human embryonic stem cells produced 2000s Human genome sequenced Gene therapy used in humans BIOTECHNOLOGY AND HEALTH 17
18 BIOTECHNOLOGY AND HEALTH Explosive expansion over the past 30 years Annual revenues are well over $100 billion More than 200 diseases currently targeted for treatment with drugs produced using biotechnology Alzheimer s disease AIDS Cancer Multiple sclerosis Diabetes Arthritis Discovery of new drugs is very expensive and time consuming RECOMBINANT DNA TECHNOLOGY A DNA molecule can be cut by enzymes and recombined with other DNA molecules Has become accepted, and ethical considerations have moved on to other issues Science has to apply itself to the rigorous standards of public scrutiny Frequently used techniques for replicating DNA: Cloning using a plasmid as vector Polymerase chain reaction CONSTRUCTING A RECOMBINANT PLASMID 18
19 CONSTRUCTING A RECOMBINANT PLASMID POLYMERASE CHAIN REACTION RECOMBINANT DNA TECHNOLOGY 19
20 MONOCLONAL ANTIBODIES Monoclonal antibodies are relevant in context of: diagnosing infectious diseases detecting harmful microorganisms in food distinguishing cancer cells from normal cells delivering chemotherapeutic drugs to cancer cells locating environmental pollutants phage display Fast, clean, safe, humane procedure required to produce large amounts of good-quality antibodies repeatedly for long periods MONOCLONAL ANTIBODIES In 1975 Köhler and Milstein developed an advanced cell culture for creating antibodies from a single cell All populations of those antibodies are exactly the same in their specificity and binding abilities The single cell from which the antibodies are produced is called a clone cell The antibodies are called monoclonal antibodies Recombinant monoclonal antibody production is based on microbial cultures, mammalian cells, and plants MONOCLONAL ANTIBODIES 20
21 MONOCLONAL ANTIBODIES PHAGE DISPLAY PRINCIPLES OF DIAGNOSTICS 21
22 SEROLOGY-BASED DIAGNOSTIC METHODS Based on an in vitro antigen antibody reaction When antibody and antigen bind in large amounts, they become insoluble and therefore visible Gruber reaction: an unknown antigen in the sample can be identified or detected by using a known antibody Widal reaction: a known antigen is used to spot an unknown antibody in the sample If we use much smaller amounts we have to develop a method that produces another type of signal (such as fluorescence) to measure binding SEROLOGY-BASED DIAGNOSTIC METHODS Precipitation A successful reaction can be seen as a white ring (ring test) If we trap antibody in a gel matrix and put the antigen sample in a well within that gel, we can use much smaller amounts. In electroimmunodiffusion the movement of the proteins in the gel is speeded up by an electric current Agglutination Works on a smaller scale and allows faster results One of the reaction partners is coupled to a bulky component such as erythrocytes or latex beads When a reaction takes place latex beads agglutination can be observed SEROLOGY-BASED DIAGNOSTIC METHODS Immunoblotting Permits the analysis of a mixture of antigens or antibodies Proteins are separated by using an electric current and then transferred to a membrane The membrane is incubated in a solution containing antibody This entire procedure is called a Western blot The antibodies bind to the antigen on the membrane The binding is detected then via a signal that can be measured The antibody can be conjugated to a fluorescent molecule, a radioisotope, or an enzyme that catalyzes a chemical reaction producing a color change or light 22
23 SEROLOGY-BASED DIAGNOSTIC METHODS SEROLOGY-BASED DIAGNOSTIC METHODS Immunoblotting Alternatively, the antibody is not labeled, but a second labeled antibody is used to bind the first antibody The secondary antibody amplifies the signal and therefore increases the sensitivity of the detection Direct immunofluorescence uses fluorescent antibodies Indirect immunofluorescence uses a secondary fluorescent antibody We can analyze antigens (on the membrane) in a sample taken from the patient, and use known antibodies for detection More commonly (e.g. HIV diagnosis) antibodies in a patient s sample are analyzed by using known antigens on the membrane SEROLOGY-BASED DIAGNOSTIC METHODS 23
24 ELISA (enzyme immunoassay/enzyme-linked immunosorbent assay) SEROLOGY-BASED DIAGNOSTIC METHODS The solid matrix for the antibody antigen binding reaction is the bottom of a well as part of a microtiter plate Smaller sample amounts are used and signal reading is automated Direct assay detects the antigen in the sample and wells are coated with known antibody Indirect assay detects antibodies in the sample (e.g. HIV diagnosis) and antigen-coated wells are used The analytical antibody is conjugated to an enzyme In a radioimmunoassay the antibody is radioisotope-labeled Dipsticks are used in health care as a very rapid EIA for quick diagnostics (at the bedside or in point-of-care analysis) DIAGNOSTIC METHODS BASED ON NUCLEIC ACIDS Identifying the pathogen is based on a nucleic acid sequence specific to a certain pathogen in a patient s sample PCR Using primers complementary to a specific pathogen gene to produce a product of the correct size DIAGNOSTIC METHODS BASED ON NUCLEIC ACIDS Hybridization Uses the binding of an oligonucleotide probe complementary to part of the pathogen DNA It can take place on a solid matrix (Southern blotting for DNA; Northern blotting for RNA) Detection of hybridization depends on the type of labeling of the probe (radiation, fluorescence, chemiluminescence, color) Rapid dipstick methods are available 24
25 DIAGNOSTIC METHODS BASED ON NUCLEIC ACIDS Hybridization High-throughput analysis is done using microarrays An array is the solid support for thousands of specified DNA segments at fixed locations. Binding of labeled probes is detected and the data are analyzed by computational methods Microarray technology allows identifying whether a certain DNA sequence is included in a sample mixture Microarray analysis can be used for sequence identification via the 6S rrna gene sequence DIAGNOSTIC METHODS BASED ON NUCLEIC ACIDS DIAGNOSTIC METHODS BASED ON NUCLEIC ACIDS 25
26 STRAINTYPING Identifying the strain (subspecies) is necessary for epidemiological studies and disease monitoring All diagnostic methods can be adapted and used for straintyping Further methods are: Antimicrobial susceptibility patterns Plasmid fingerprinting Restriction endonuclease analysis of genomic DNA Phagetyping DIAGNOSTICS THE FUTURE We need: More sensitive technology (requiring a smaller sample) Results being available faster and with as little manual effort as possible High specificity, consistency, and reliability DIAGNOSTICS THE FUTURE Bioinformatics deals with the data and information overload Large-scale analysis of DNA (genomics) and RNA (transcriptomics) is only the beginning The presence of functional protein in the cell is crucial and cannot be deduced from genomics and transcriptomics Proteomics analyzes all the proteins expressed and modified in a cell and their relationships to one another and to the host organism. High-resolution twodimensional electrophoresis and protein characterization are widely used 26
27 DIAGNOSTICS THE FUTURE Proteomics aid Searching for diagnostic markers Studying virulence factors Identifying suitable antigens for vaccine development Developing and validating targets of new anti-infective compounds Investigating the epidemiology and taxonomy of human pathogens Identifying new novel pathogenic mechanisms and mechanisms of drug resistance DIAGNOSTICS THE FUTURE Biosensors and nanotechnology open exciting opportunities for further advancement in the detection and quantification of biological molecules This moves modern molecular diagnostics from hospital laboratories to the patient Electronic noses (certain sensors) can detect volatile organic compounds directly from clinical materials Magnetic immunoassays and bio-barcode assays can detect both DNA and protein thousand times better than the best ELISA Nanocantilevers can detect a single E. coli bacterium 27
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