Module 3 Essay Plans Flashcards
Describe examples of first, second, and third generation sequencing technologies, and give examples of how it can be used in the context of infectious disease
1st - Sanger Sequencing:
Involves fragmenting and amplifying DNA, then mixing it with ddNTPs which don’t have a free 3’ OH, and so terminate polymerisation. The different fragments formed are then separated by electrophoresis. This technique is quick and very accurate, but is far more expensive than other methods.
2nd - Pacific Bio
A real time technique that is fast and cheap with a long read length, but is inaccurate
2nd - Illumina
Very accurate and cheap, but slow and has a short read length
3rd - Nanopore:
Hollow transmembrane proteins are inserted into a membrane of high electrical resistance with a current applied across it. As a single strand of DNA passes through the pore, the different bases cause distinct changes in the current, which are detected in real time. The issue with this technology is its comparatively low accuracy: 80-85%. However the technology can be very compact and easy to transport, and so could be used to identify DNA rapidly in locations without labs.
Gene sequencing with the intent to identify bacteria often focuses on identifying variable portions of 16s RNA, part of the ribosomal RNA which is present across all bacteria. Focusing on the variable region allows identification of the species. .
Next generation sequencing can be used to:
Identify microbiome components:
Microbiome research is a rapidly expanding field of study, as mircobiome chnages have been liked to pathologies in every body system. Sequencing of the bacteria in an individual’s microbiome is useful for research, and may in the future be used to guide treatments
Identify causative agents:
E.g. analysis of sputum samples from CF patients found S. Contellatus which had not been found in cultures because it doesn’t grow in culture (Sibley 2008). Sequencing has also been shown to be a faster and cheaper alternative to standard lab diagnosis for TB
Identify new pathogens
E.g. when an outbreak of an unknown pathogen occurred in a USA clinic, RNA was isolated from patient samples, known pathogenic RNA and human RNA was removed and a new virus (Arena virus) was identified (Palacios 2008)
Identify antibiotic targets:
E.g. (Andreis 2005) identified the first new TB antibiotic for 40 years. TB strains were grown with antibiotics to reveal resistant strains. Resistant strains were sequenced and the gene common to all of them was identified (coded for part of F0 subunit of ATPase), leading to the creation of a new drug for resistant TB.
Identify essential genes
This helps to develop therapies against drugs or determine the reason for pathologies (e.g. Langridge 2009 found S. typhi genes for surviving in bile salt)
Analyse disease outbreaks
E.g. Haiti cholera epidemic strain was traced back to region of Nepal, where UN peacekeepers had come from. Allows monitoring of strains for resistance etc. during outbreaks
Design growth mediums
Identify new antimicrobials
Describe different methods for diagnosing TB in children and their advantages and disadvantages. Describe the challenges of treating children for TB, and recommendations for management
Tuberculin skin test
Involves subcutaneous injection of TB antigens and monitoring for sensitivity response. It requires some technical skill. It is not specific, and can be falsely positive because of: environmental mycobacteria presence, BCG, or latent disease. It is not sensitive and can be falsely negative in: early disease, severe malnutrition, acute viral infections (measles, whooping cough), children, and compromised patients.
Microscopy
Staining with Ziehl-Neeson reveals acid-fast bacili (consequence of hydrophobic mycolic acid cell wall). However it takes 4-6 weeks to culture samples, requires a prohibitive amount of resources, and sufficient samples of sputum or gastric washings are hard to get, and children are often paucibacillary, making this test insensitive. Although the BACTEC MGIT system is faster (9-16 days) it is expensive.
GeneXpert
The GeneXpert machine purifies and concentrates TB samples and amplifies Mtb DNA. Within 2 hours the machine can give a diagnosis, and detect rifampicin resistance, and the test is sensitive and specific. Though the machine is expensive, each separate test is relatively cheap
IGRA
Interferon gamma release assays measure TB infection by analysing the host immune response in a blood sample that is then stimulated with antigen. It is essentially the same principle as a tuberculin test, but is more MtB specific. However it is technically challenging and expensive, and carries ethical issues RE venepuncture in children. It also does not discriminate between infection and disease, and infection only develops in ~10% of infected people.
MODS
Microscopic observation drug susceptibility testing is microscopy of decontaminated sputum samples cultured in broth for 8-15 days, which is faster than using solid media. Mtb forms unique cords in culture which can be observed. This method allows for diagnosis and testing for drug resistance. It is cheap, low-tech, fast, sensitive, and specific.
PCR
PCR can be used to amplify Mtb-specific regions of DNA. It is specific, but sensitivity varies between commercial and in house machines. Can be used to identify serotype
LAM urine test
An easy point of care, 30 minute, very cheap ($3.50) test which is useful for HIV+ adults with suspected TB, as its sensitivity increases the lower the CD4 count is. It works by detecting lipoarabinomannan from TB cell walls in urine, but has poor sensitivity in children, and does not differentiate disease from infection
Difficulties in treating children:
Need frequent dose adjustment due to growth
Compliance issues - directly observed therapy course
Have to treat without culture confirmed diagnosis because children are paucibacillary and tests aren’t sensitive enough
Few drug studies have been done in children, so there is little evidence to base treatment on
Liver: body ratio is higher so metabolism and excretion of drug is different
Children are more vulnerable to active infection and extrapulmonary manifestations
Initial presentation is easy to mistake for other childhood infections
Management recommendations Prevent if possible Avoid contact with infected adults Screen high risk groups Maintain general good health and nutrition Don't delay treatment if TB is suspected Ensure adherence through support
Describe how in vivo and in vitro models can be used to study virulence factors in bacteria
In vitro models are the mainstay of pathogenecity research, as they are the easiest and simplest way to study pathogens. The main issue is that the cultured cell lines do not accurately replicate the in vivo environment. This issue can be addressed in several ways: multiple cell types can be assembled into a network, in an attempt to replicate the multiple tissues of an organism. Furthermore, in vivo environments can be simulated e.g. by introducing flow over endothelial cells to simulate a blood vessel, or by using a vacuum pump to simulate lung expansion with epithelial cells. Cells can also be grown on mediums designed to replicate in vivo conditions e.g. collagen, fibronectin. Explants are tissue samples taken from living organisms and transplanted to media, and they help to replicate the in vivo phenotype, but are difficult to obtain, and may be altered in the process of their removal.
Animals:
Rodents are most commonly used: they are cheap and easy to obtain, and it is easy to introduce gene knockouts or novel genes. However laboratory rodents are inbred, resulting in expression of a limited range of MHC alleles. As a result, their immune response will not always mimic that of humans particularly well, and they are not considered good models for studying intracellular pathogens.
Other animals are used, and the species is selected depending on the disease being studied (e.g. chinchillas for otitis media, guinea pigs for TB, primates for HIV, though consent is rarely obtained). Although specific pathogens are best studied in certain animals in order to approximate human immune response, there is always a risk that animal research will not be applicable to humans
In order to evaluate the severity of disease in an animal model, the infectious outcome must be decided e.g. ID50 vs. LD50. ID50 is the number of bacteria necessary to infect 50% of animals, whereas LD50 is the number of bacteria necessary to kill 50% of bacteria. LD50 is difficult to obtain ethical consent for. These measures are useful, but can be misleading, as some more lethal pathogens require a higher infective dose than safer pathogens (e.g. cholera vs. shigella).
Single-tagged mutagenesis in animal testing can be used to determine essential virulence factors that may be used as therapeutic targets. Transposons are used to inactivate one particular virulence gene, and animals are exposed to the pathogen. Later, the pathogen is extracted from infected animals and compared to the pathogen given to the animal. Strains that are not seen after infection are then known to have lost their virulence due to the mutation, which can then be targeted therapeutically.
Humans:
The ideal host for modelling disease as it is obviously the most accurate. However human testing is ethically tricky and requires sophisticated facilities to contain the pathogen (e.g. total control air handling, en-suite bathroom). Furthermore the recruitment and follow-up is logistically challenging
Conclusion
Decide based on cost, relevance of model to pathogen, question you’re answering, reproducibility of results in humans
Define immune tolerance and discuss the mechanism of central and peripheral tolerance. Which are the most important mechanisms of immune tolerance for treatment? Discuss allergen immunotherapy and the mechanism involved.
Tolerance refers to a state of immune functional unresponsiveness to an antigen. Failure to develop tolerance may lead to atopy, or to full allergic or autoimmune disease.
Central tolerance is regulated in the thymus, where T-cells develop. T-cell receptors are generated via VDJ recombination, producing receptors against all conceivable antigens, but t-cells must be screened to make sure they work, and don’t target self. T-cells first undergo positive selection in the cortex to confirm that they can bind to MHC. They then undergo negative selection in the medulla to confirm that they are not self-reactive. Presence of the AIRE transcription factor causes transcription of self-antigens from various organs across the whole body within the thymus. T-cells that bind to any self-antigen undergo clonal deletion in the medulla. This induces tolerance of the body’s own tissues.
B-cells mature in the bone marrow, and undergo an analogous process. However, there is less deletion in this process, and a higher chance of autoreactive B-cells being released into circulation, though this is less severe than having autoreactive t-cells.
Peripheral tolerance is the control of over-reactive lymphocytes in the circulation, and can be induced through exposure to the antigen, generally via the gastrointestinal tract. High-dose exposure in the gut usually results in either clonal deletion of the reactive T-cell (via CD95 activation by CD95L on APC) or anergy (due to a lack of CD28 co-stimulation). Contrastingly, low dose exposure causes temporary T-cell suppression via release of IL-10 and TGF-beta by Treg cells, though this can be disrupted leading to a loss of tolerance. Treg cells are a distinct CD4+ subpopulation which may be created in the thymus or induced in the periphery. Treg cells are crucial for normal immune function, as illustrated by IPEX syndrome: a rare X-linked condition caused by FOXP3 transcription factor dysfunction leading to Treg dysfunction and subsequent autoimmunity.
Tolerance can be affected by Ig status and the gut microbiome. Development of tolerance in the gut requires uptake of antigens via IgA; if there is insufficient IgA and the antigen is instead taken up by IgE, there is a greater chance of allergy forming instead of tolerance. IgA and Treg cell responses are both impaired if there is insufficient bacterial input, as specific bacteria prime dendritic cells to induce Treg cells.
Other determining factors in the development of allergy or tolerance include dose (intermittent low dose favours allergy, regular high dose favours tolerance), route of exposure (gut promotes tolerance as proteins are partially degraded, inhalation or skin contact promotes allergy hence eczema is linked to allergy as barrier is impaired). Exposure to likely allergens early in life through feeding has been shown to decrease the rate of allergies (EAT study, Israeli baby peanut snacks)).
Immune tolerance therapy generally aims to induce tolerance of an antigen through regular exposure over several months. This has been shown to be safe and effective in reducing symptoms and medication use for grass pollen (SLIT), and severe systemic insect venom allergies (though there are doubts over its cost-effectiveness).
Immune tolerance therapy for food allergens carries more concerns. There are adverse reactions in ~90% of patients, with ~10% of patients needing to administer adrenaline. Adverse events are particularly unpredictable, and sometimes occur at previously tolerated doses. Oral immune tolerance therapy has been shown to only be successful in 50-60% of patients, and only half of those remained tolerant. Induction of tolerance to food allergen takes time, and so there are also compliance issues.
Compare and contrast different ways of diagnosing allergic disease and mechanisms
IgE allergy is much easier to diagnose than non-IgE-mediated allergy, as it presents with acute…
Skin tests:
Either a skin prick test or an intradermal test can be done (the latter being trickier and more painful, but also more sensitive). They produce a ‘wheal and flare’ reaction due to mast cell degranulation. Both are quick, cheap, easy (though excessive force or insufficient entry into the skin can cause false positive or negative), and give fast results. They have a high negative predictive value, and a fair positive predictive value, but carry a small risk of causing a systemic allergic reaction. The skin tests also have a variable response depending on time of day (circadian rhythm), season, and potency of the allergen (e.g. drug and venom allergies may require more sensitive intradermal test). Furthermore, skin tests are unsuitable in patients with dermatographism, severe dermatitis, recent anaphylaxis, or who are taking anti-histamines.
Blood Tests:
Specific IgE: a fluorescent antibody test using the allergen absorbed onto a cellulose sponge measures which allergens the patient has produced IgE against, then fluorescent anti-IgE antibodies bind to them. It’s more expensive, slower than skin tests, has a lower negative predictive value, and there are ethical and practical issues around taking blood in children. Blood tests are useful in cases where the patient has severe dermatitis, or recent anaphylaxis, or is taking anti-histamines. Basophil activation test can be done, but isn’t really.
In vivo test:
40% of young adults will have a positive skin test to some common allergens, but only ~20% develop symptoms if challenged (i.e. positive predictive value is not high). Therefore a more relevant test is to challenge the patient with the allergen and monitor them for symptoms. This is done with oral food challenges, drug provocation tests, dermatitis patch tests etc. These tests are riskier than skin or blood testing, and so require access to a properly trained clinician, and full equipment to deal with anaphylaxis.
Describe known causes of primary immunodeficiency, and how they have contributed, or may contribute, to management of ID
Primary immunodeficiency refers to immune dysfunction caused by a genetic disorder, as opposed to secondary immunodeficiency which is the malfunction of a previously healthy immune system e.g. due to burns, AIDS, or medication. Study of primary immune deficiency is helpful as it highlights crucial immune system genes in the normal response to infection.
Defects in different aspects of the immune response lead to different types of immune dysfunction: T-cell defects (e.g. diGeorge syndrome) lead to intracellular pathogen susceptibility; B-cell defects (e.g. X-linked agammaglobulinaemia) lead to extracellular pathogen susceptibility; complement defects increase vulnerability to bacterial infection (especially N. meningitidis); and neutrophil defects increase vulnerability to pyogenic bacterial infections.
SCID refers to a rare genetic condition caused by disrupted development of B and T cells. There are multiple mutations which can cause SCID, most commonly an X-linked mutation in gamma chains which leads to non-functional interleukin receptors which cannot signal B and T cells to activate and multiply. Other mutations may cause SCID: e.g. RAG1/RAG2 mutations which interfere with VDJ recombination and hence impair lymphocyte receptor manufacture.
Genetic pre-disposition to invasive pneumococcal disease:
IRAK-4 or MyD88 mutation disables function of all TLRs except 3 which leads to deficiency in TNF and IL-1 signalling. Children with these mutations developed severe invasive disease (pneumonia, sepsis, meningitis) from strep pneumoniae. This demonstrated that IRAK-4 and MYD88 were key for pyogenic bacteria defence, but fairly redundant regarding viral, mycobacterial, or fungal defence
Genetic predisposition to herpes simplex encephalitis:
HSV usually causes minor disease (e.g. cold sores) and then becomes latent inside nerve ganglions, occasionally reoccurring. In patients with herpes simplex encephalitis, UNC93B was found to be mutated. UNC93B is an ER resident protein required for TLR3 expression. TLR3 activates interferon production, hence mutation led to uncontrolled herpes.
Discuss the evidence for environmental factors in the development of allergic disease (40%). Using one allergic disease (e.g. asthma, eczema, allergic rhinitis) discuss the genetic factors in the development of allergic disease (30%). Explain how the environment and genetics interact in the development of allergic disease (30%)
Rates of allergic disease have risen steeply over the last 60-70 years, with NHANE surveys reporting a doubling in the rate of sensitivity to one or more allergens over less than 20 years. This increase in allergy is often attributed to an increasingly hygienic lifestyle, which minimises contact with various antigens. This is supported by the PARSIFAL study, where growing up on a farm, and engaging in a lifestyle free of pasteurised food and antibiotics was found to be protective against allergic disease.
This is further supported by observations that in Germany before the fall of the Berlin wall, the opulent West side saw greater incidences of asthma and hayfever than the relatively poor East. The difference in wealth is theorised to have led to a less hygienic environment in East Germany, resulting in greater exposure to de-sensitising antigens. Finally, one study examined rates of atopic disease in villagers in rural Poland at the time Poland joined the EU, and 9 years after. During this time agriculture underwent mechanisation, and fewer villagers came into contact with the farm environment. Over this time, rates of atopic disease within the village population increased significantly.
The incidence of eczema has risen in line with other allergic disease: surveys of Aberdeen schoolchildren between 9-11 in 1964 and 2014 revealed rates of 3% and 29% respectively. Eczema is characterised by a triad of skin barrier defects, skin innate immunity defects and enhanced response to food and inhalant allergens.
A key gene implicated in the pathophysiology of eczema is filaggrin, which is an epidermal barrier protein. Loss of function of filaggrin leads to leaky skin which becomes dehydrated and thus a less effective barrier. Filaggrin is strongly associated with eczema, and increased severity eczema, as well as allergic rhinitis and food allergy. This association is thought to be due to exposure to allergens vis the impaired skin barrier, which leads to allergy instead of tolerance.
Dysfunction of SPINK5, an anti-protease gene, and Claudin-1, a tight junction gene, are both also associated with eczema. Dysfunction of either gene leads to impaired skin barrier function, and uptake of allergens leading to Th2 allergic responses.
Discuss how 16srRNA research has contributed to PID
16srRNA is a component of the 30s subunit in bacterial ribosomes. It is found in all bacteria and contains both highly conserved and variable regions. Therefore 16srRNA sequencing can be used to identify bacteria by designing complementary primers to conserved regions to sequence the variable regions. Though this can identify the genus, it cannot always identify the species of bacteria.
16srRNA was used in the neonatal microbiota study where faecal samples were collected and bacterial DNA extracted. 16srRNA was amplified by PCR and sequenced. This led to the discovery that babies who developed necrotising enterocolitis had C. perfringens and there was some expression of beta2 toxin which was not found in healthy babies. The presence of C perfringens was associated with earlier onset, more severe disease, and higher mortality
Discuss how GWAS research has contributed to PID
GWAS is sued to analyse DNA of subjects with a certain disease for genetic markers, usually single-nucleotide polymorphisms. This may demonstrate genetic associations with disease, but cannot prove a casual link, and can’t be used with Mendelian disorders (too rare).
GWAS identified IL-33 as an asthma-associated gene
Discuss how Proteomics research has contributed to PID
Proteomics is the study of protein production, usually via electrophoresis and mass spectometry. Proteins are promising biomarkers as they provide the most accurate possible representation of cell activity. Proteomic studies are time and cell specific, and allow identification of protein isoforms which provide evidence about post-translation modifications.
Protein biomarkers include hCG, Ca-125, and CRP. In research, proteomics has identified 4 protein markers for TB. These markers are sensitive and specific, which is particularly important given how poor the current mainstream options for TB diagnosis are. Monitoring of protein production may in the future provide insight into key proteins for pathogens, that may lead to antibiotic or vaccine development, and a better understanding of pathogen-host interactions.
Discuss how microarrays research has contributed to PID
Microarrays are known series of nucleic acids immobilised on a solid substrate. Test samples are washed over the microarray and complementary sequences hybridise and stick. Gene expression may be quantified by fluorescence. This allows the measurement of expression of large numbers of genes simultaneously, and allows comparison of the DNA sequences between individuals.
Microarrays are used in GWAS to identify genetic markers that are associated with specific diseases. They may also be used in combination with 16srRNA to identify bacteria, or to provide cheap, easy, fast, point-of-care tests for biomarkers of paediatric diseases (e.g. TB). They may also be used to study the immunopathology of host responses to pathogens
Microarrays were used to carry out transcriptomics to examine the link between meningiococcal sepsis and myocardial failure. Specific genes upregulated in sepsis were identified, and it was found that IL-6 depressed myocardial function. Implications in blocking IL-6 for treatment or prevention.
Discuss how Transcriptomics research has contributed to PID
Transcriptomics refers to measurement of the total mRNA in a cell or organism, and so monitors changes across a whole genome simultaneously. This is compared to control groups to provide gene expression profiles in response to specific stimuli.
Transcriptomics provides insight into immunopathology by showing which genes are activated, and gene expression patterns can be used to classify diseases.
Some transcription changes are very specific to pathogens, and so transcriptomics can be used to differentiate between organisms with similar clinical presentation.
Transcriptomic analysis can highlight virulence factors which can be targeted in treatment, as they will be clustered in one area due to horizontal gene transmission
Transcriptomics is also promising for distinguishing viral from bacterial disease via 2 key transcripts that have been identified and could form a ‘traffic light’ test for antibiotic use.
