Treatment of Infections Flashcards

Question

Inhibitors of protein synthesis

Answer 1

- Bacterial & mammalian ribosomes differ in structure, these difference are used to inhibit bacterial protein synthesis. - Two sub units difference. Bacterial 50S, 30S Mammalian 60S 40S - Within subunits the size of ribosomal RNA is also different, 23S vs 26S

Answer 2

Inhibitors of protein synthesis: Aminoglycosides (e.g. gentamicin) Reversed for hospital settings, only for serious infections Serious side effects, requires therapeutic does monitoring Bind irreversibly to 30S subunit, bactericidal

Answer 3

THREE phases of translation: 1) Initiation: - The ribosome assembles around the template mRNA. - The first tRNA is attached at the start codon. 2) Elongation: - The tRNA transfers an amino acid to the mRNA corresponding to the next codon. - The ribosome then moves (translocates) to the next mRNA codon to continue the process, creating an amino acid chain. 3) Termination - When a stop codon is reached, the ribosome releases the polypeptide.

Answer 4

- Tetracyclines reversibly bind to acceptor site in 30S subunit - Prevents tRNA binding to mRNA at acceptors site, no proteins synthesis - Reversible binding means only bacteriostatic - Also binds to our own ribosomes, but bacteria accumulates it within their cells, hence it’s a selective drug - Must be aware of side effects when used - Targets 30S subunits

Answer 5

4 methods in which they work 1. Bind to region in acceptor site, inhibits binding of tRNA to acceptor site 2. Prevents initiation complex forming (stage where 2 subunits combine around tRNA & mRNA) 3. Cause codon misreading along mRNA, produces non-functional proteins 4. Can cause membranes to become permeable – allows aminoglycosides to accumulate in cell & increase cidal effects

Answer 6

- Mutations don’t occur often, but bacterial colonies contain millions of cells. - Some mutations may confer resistance to antibiotics - Under normal conditions this may slow growth, but when treated they survive & divide, producing a resistant population - Bacteria continue to gain resistance to antibacterial drugs

Answer 7

Plan to reduce AMR: - Reduce need for & exposure to antimicrobials e.g. sanitation - Optimizing antimicrobial use e.g. Antibiotic stewardship - Investing in innovation e.g. new antimicrobials

Answer 8

Noncompliance Aging population Poor infection control in healthcare settings Industrial use and production of antibiotics Lack of development of new drugs

Answer 9

- Intrinsic resistance is a bacteria’s natural resistance to an antimicrobial. - All strains of one particular species will be resistant to a certain class of antimicrobial. - e.g. Mycoplasma sp are an intracellular bacteria -live inside other cells- & have NO cell wall – has intrinsic resistance to classes of antibacterial which target the cell wall including the beta lactams.

Answer 10

Acquired resistance is when bacteria develop resistance, only some bacteria in a species will be resistant. There are 2 type of acquired resistance: - genetic (irreversible) - phenotypic (reversible)

Answer 11

- phenotypic (reversible): resistance achievable without genetic alteration. - Phenotypic resistance is associated with specific processes such as growth in biofilms, as the extracellular matrix gives antibiotic resistance, which is lost when no longer in the matrix. - e.g. E.coli grows in a biofilm

Answer 12

Genetic resistance (irreversible): Resistance is transferred to daughter cells. Genetic resistance can develop through: - acquiring of mutations in the chromosomal DNA - Acquiring new DNA such as a plasmid through bacterial conjugation, which can occur between species - Insertion of genetic material into the bacteria e.g. MeCA inserted into MRSA

Answer 13

A plasmid can contain multiple antibiotic resistance genes. If a bacteria acquired this, it would become resistant to all the drugs encoded by it. Conjugation allows for intra & inter species transfer, meaning resistance spreads easily.

Answer 14

1 -inactivate or modify the drug itself. 2 -alter the drugs target site e.g. altering drug binding site but without effecting how the enzyme functions. If the drug can’t bind it can’t work. 3 altered transport – either prevent the drug entering the cell Or remove the drug from the cell before it can reach an [effective]

Answer 15

- Bacteria can make beta lactamase, which hydrolyses beta lactams, rendering them inactive. - Enzymes can be encoded on bacterial chromosome e.g. S.aureus, P.aeruginosa - Commonly be encoded on a plasmid, but can also be chromosol.

Answer 16

- Many different beta lactamases encoded on plasmids, each work differently - Individual beta lactamases won’t inactivate each type of beta lactam antibiotic, differing in spectrums of activity. - Some are limited. E.g. only penicillin - e.g. BLA TEM1 & BLA TEM2 - TEM1 is the plasma encoded beta lactamase gene conferring resistance to Ampicillin in E.coli

Answer 17

Extended spectrum ESBLS have broader spectrums of activity – don’t hydrolyse penicillin, inactivate many other beta lactams. e.g. BLA CTX-M (some) BLA NDM-1(all beta lactams), Bacteria with these genes are “superbugs,” due to limited treatment options.

Answer 18

- Gram negative bacteria accumulate beta lactamases intracellularly - Gram positive bacteria excrete the beta lactamases from the cell - Gram-negative bacteria are more effective at disabling our beta lactam antibiotics.

Answer 19

We prevent inactivation by beta lactamases by using beta lactamases inhibitors. The inhibitors don’t have antimicrobial activity but inactivate beta lactamases. Given with beta lactam can extend their life. e.g. clavulanic acid (beta lactamase inhibitor) given with amoxicillin, creating Co-amoxiclav.

Answer 20

Beta lactam antibiotics have side chains which make them resistant to some beta lactamases. e.g. Flucloxacillin, Isoxazoyl on a side chain inhibits some beta lactamases activity.

Answer 21

- Altering target sites can give resistance to beta lactam drugs. - The target site is PBP (Penicillin Binding Protein) outside the bacteria’s cytoplasmic membrane, there are multiple PBPs. - The beta lactams bind to the PBP, changing its’ structure, preventing beta lactam binding, meaning it cannot inhibit cell wall biosynthesis. - The change doesn’t affect PBPs enzyme function, just prevents binding. - Bacteria with altered PBPs can occur due to gene changes. e.g. MRSA, where MecA changes PBP2 to an altered target site.

Answer 22

Gram-negative bacteria reduce the size & number of porins, decreasing permeability & prevents uptake. This prevents antibacterial entering the cell & reaching their target.

Answer 23

Vancomycin is a glycopeptide, inhibiting - the crosslinking of peptides by binding to the side chains and preventing the removal of D-alanine (final crosslinking step). - formation of the glycosidic bonds by binding to the terminal amino acids of the peptide side chain. Vancomycin no longer forms stable complex, hence altered target site. Can still bind, with 4 H bonds, not 5, reducing binding affinity, becoming unstable & less effective, hence the bacteria becomes resistant. Can get resistance through chromosomal & plasmid encoded resistance e.g. Van A resistance.

Answer 24

Van A resistance alters terminal amino acid on the peptide side chain on the peptidoglycan monomer, from alanine to a lactate. VanA plasmid causes resistance in enterococcal bacteria, causes UTI, blood stream infections & endocarditis. VRE- Problematic as Vancomycin is first choice if penicillin allergy, & VRE strains resist both Vancomycin & its replacement, Telicoplanin.

Answer 25

Bacteria produce aminoglycoside modifying enzymes, which modify & cause inactivation. Classified into 3 types based on how they modify the aminoglycoside: 1. Acetylases – adds acetyl groups 2. Phosphorylases – adds phosphate groups 3. Adenylases – adds AMP groups These modifying enzymes are usually expressed from plasmids

Answer 26

Fluoroquinolones target DNA Gyrase &/or Topoisomerase IV, which are involved in untangling supercoiled DNA, & separating the interlinked daughter chromosomes. Resistance occurs by altering of the drugs target, such as mutation, inhibiting binding, not action.

Answer 27

- Tetracycline’s inhibit protein synthesis, so to be effective they enter the cell & reach the cytoplasm. - However, both Gram-negative & Gram-positive bacteria have efflux pumps across their membranes & these pump tetracycline out of the cell, preventing the drug from reaching [the] required to inhibit protein synthesis. - Efflux pumps can give resistance to a single type of antibiotic, or multiple. - The tetracycline pump is a specific pump. It only works in the presence of Tetracyline (inducible pump), & it’s encoded by a plasmid. Inducibility means energy isn’t expended unnecessarily. - Pseudomonas have multiple efflux pumps, causing it to be difficult to treat, - The efflux pumps can be expressed chromosomally, with basal expression, so lower amounts are pumped from the cell, giving a low-level intrinsic resistance. - Mutations can increase upregulation of these pumps, giving a stronger effect (acquired resistances).

Answer 28

- Different efflux pumps can produce multi drug resistance, & if they become upregulated, they can pump out multiple different types of drug in one go. - 2 classes of efflux pump are particularly associated with multi drug resistance: 1. called MFS associated with gram positive bacteria 2. We have RND pumps expressed in gram-ve bacteria e.g. RND It’s the ACR AB/ TolC pump in E.coli, a chromosomally encoded pump & it usually confers a low level of intrinsic resistance. If it's upregulated it confers a high level of resistance to multiple antibacterial agents in one go.

Answer 29

- AMS is the approach & monitoring of antimicrobials to ensure future effectiveness. - Aims to cause least harm to patient & future patients. - Assess and consider the appropriate use of antimicrobials.

Answer 30

- Blood tests including: - White Blood Cells (WBC) - bacterial infection typically causes neutrophilia - inflammatory markers (C-Reactive Protein, CRP) - Blood cultures - Local sites e.g. chest X-ray, urine culture, sputum culture

Answer 31

Infection can present +/- fever +/- local symptoms - Older people may present with a change in mental status or decline in function, often without fever. - Children with a serious infection may compensate well at first, so there is a risk of missing a serious ill child.

Answer 32

Sore throat: Usually self-limiting to a week Treatment: - Drink fluids - Ibuprofen, paracetamol - Medicated lozenges (local anaesthetics, NSAID, antiseptic) - Avoid hot drinks

Answer 33

Group A streptococcus (GAS) is the commonest bacterial cause of sore throat. Treat with antibiotics to prevent acute rheumatic fever, reduce severity and prevent transmission. Use phenoxymethylpenicillin, if penicillin allergic use clarithromycin. FeverPAIN score /5 determines likelihood of Streptococcal infection Fever (> 38°) +1 Purulence (pharyngeal/tonsillar exudate) +1 Attend rapidly (within 3 days of onset) +1 Inflamed tonsils +1 No cough or coryza (cold symptoms) +1

Answer 34

Epiglottitis = inflammation of the epiglottis. Symptoms can include: - Severe sore throat - Stridor (harsh sound from obstructed airway) - high fever - Dysphonia: muffled or hoarse voice - Drooling - Dysphagia: difficulty or pain swallowing - Distress: Tend to sit upright/leaning forward, breathing carefully.

Answer 35

- Infectious mononucleosis (glandular fever) – suspect if more than a few days, adenopathy, splenomegaly - Retropharyngeal or peritonsillar abscess (quinsy) – fever, neck pain, muffled voice +/- trismus (restricted mouth opening), +/- displaced uvula, enlarged, displaced tonsil - Signs of scarlet fever (Group A streptococcus) Sandpaper rash, strawberry tongue

Answer 36

Lemierre syndrome: thrombophlebitis of the jugular vein. Typically in people with systemic inflammatory response syndrome (SIRS) or sepsis.

Answer 37

Haematological disorders: Leukaemia: ulceration and haemorrhage of the mucous membrane of the pharynx may occur. For more information, see the CKS topic on Haematological cancers - recognition and referral. Aplastic anaemia: sloughing and ulceration of the mouth and pharynx may occur.

Answer 38

Kawasaki disease — may occasionally present as sore throat without exudate. The cause of Kawasaki disease is unknown. The majority of cases occur in children under 4 years of age. Kawasaki disease is diagnosed by the presence of either five of the six main symptoms, or four main symptoms if coronary aneurysms are identified by echocardiography. The six main symptoms are: - Fever lasting 5 days or more. - Bilateral conjunctivitis. - Changes in the lips & oral cavity (dryness & fissuring of the lips, redness of oral cavity & pharynx). - Cervical lymphadenopathy. - Polymorphous rash (a combination of morbilliform, scarlatiniform, urticariform, or erythema multiforme-like rash). - Changes in the extremities (reddening of the palms and soles, desquamation of the skin, & oedema of the hands and feet).

Answer 39

Diphtheria: consider if oropharyngeal membranes appear grey/green, and/or the person has not been vaccinated. Take throat swabs for culture to confirm the diagnosis.

Answer 40

Measles: suggested by conjunctivitis, rhinitis, cough, and characteristic maculopapular rash in a person who is non-vaccinated. Koplik spots (bluish-white, raised lesions on an erythematous base on the buccal mucosa) are characteristic.

Answer 41

HIV-1: sore throat may be a feature of primary HIV infection and occurs after an incubation period of 3–5 weeks, with myalgia, arthralgia, lethargy, and in some people a non-itchy maculopapular rash. Lymphadenopathy develops a week later.

Answer 42

Oropharyngeal cancer: suggested by hoarseness, dysphonia, sore throat, difficulty swallowing, and neck mass or adenopathy.

Answer 43

Behcet's syndrome — suggested by vesicles on pharynx, genital ulcers, skin and eye lesions.

Answer 44

Sepsis is a life-threatening reaction to an infection, which happens when the immune system overreacts to an infection & damages your body's own tissues & organs. It can be triggered by an infection in any site: half of sepsis cases are triggered by pneumonia.

Answer 45

PROS: Promote an evidence-based and proportionate approach to managing risk and prevent indiscriminate use of antibacterials. CONS: Low risk ≠ no risk. Only capture patient condition at a moment in time: may deteriorate. May miss other conditions.

Answer 46

Advise to seek follow-up if: Symptoms have not improved — so that antibiotic treatment can be initiated and/or alternative diagnoses considered. URGENTLY IF it becomes difficult to swallow saliva or liquids, if any difficulty in breathing develops, or if there is any one-sided neck or throat swelling — so that the need for hospital admission can be reassessed.

Answer 47

Categories of test. 1) Identification of microorganisms by isolation & culture Microscopy, Culture & Sensitivity (MC&S), Biochemical tests, 2) Identification of a specific microbial gene or product. (Molecular methods) PCR , MALDI-ToF-MS, 3) Detection of specific antigens or antibodies to a pathogen. ELISA, Lateral flow.

Answer 48

Good Quality Samples depend on: - Collecting specimens at the optimal time - Collecting appropriate samples: - Taking specimens correctly and with minimal contamination - Getting samples to the laboratory with the minimum of delay - Provide relevant clinical information. - Ideally sample is taken Prior to the start of antimicrobials. - Poor sample = poor results

Answer 49

- Microscopy Cell Morphology & staining - Culture Isolation of pure sample –Streak plate Differential or selective media - Identification Biochemistry assay - Sensitivity MIC & Disc test

Answer 50

Staining techniques: - Gram staining - most common - Acid fast staining of Myobacteria - Other – Specialized strains for identifying specific bacteria - Microscopy & staining can provide a rapid indication of the type of infection. - It can distinguish between a Gram-positive cocci or a Gram-negative rod.

Answer 51

Pink (counter stain): Gram-negative Purple (Crystal Violet): Gram-positive

Answer 52

- Thick waxy outer layer makes them resistant to many common staining techniques. - Stain is forced in by heat: Heat helps the stain penetrate the waxy cell wall. - Exposed to strong acid – stain removed: The stained sample is then washed with an acid-alcohol solution. Non-acid-fast bacteria lose the stain during this step. - Mycobacteria can’t be decolorized = ACID FAST: Mycobacteria retain the red stain despite the acid wash, because of their thick cell wall. - -- Appear red under the microscope, while non-acid-fast bacteria appear blue or green from the counterstain. - This staining technique is essential for diagnosing diseases like tuberculosis and leprosy, caused by Mycobacteria.

Answer 53

Positives - Rapid - Cheap - Requires experience. - Confirms bacterial infection - Can rule some pathogens out Negatives - Need to have bacteria in sample you stain. - Don’t get definitive answer. - Doesn’t give sensitivity

Answer 54

Streak plates (Bacteria) - Used to produce single colonies – most follow-on tests only work on a single species - Less effective if infection caused by multiple pathogens, or sample contaminated Type of media: - Variety of nutrients, differential or selective media, aerobic & anaerobic conditions used dependent on suspected pathogen - Blood cultures: grown in liquid in aerobic & anaerobic conditions- then streaked out onto plates. Viruses: - Cultured in Tissue culture cell lines

Answer 55

Nutrient Agar- Not so useful in clinical lab. Differential media Example: Blood agar - Most types of bacteria grow- - Differential lysis of the red blood cells (RBC). (Haemolytic) - Alpha partially lyse RBC, -greenish colour - Beta completely lyse RBC, -area of total clearing - Gamma do not lyse RBC -media unchanged.

Answer 56

Selective & Differential media - Example: MacConkey agar - Selective: Contains bile salts/ crystal violet. Inhibits Gram-positive. Selects Gram-negative bacteria - Differential: Lactose & pH indictor- Colour change - Lactose fermenters- metabolise lactose causes acidic agar, meaning pH indictor shows colonies PINK. - Non-lactose fermentor- not acidic- Colonies colourless.

Answer 57

Disc diffusion method (Kirby Bauer) - Single colony used to make bacterial lawn - Paper disk impregnated with antimicrobial - diffuses into agar - Zone of inhibition measured (mm) - Qualitative: Resistant, intermediate, or sensitive. - Compared to standardized table

Answer 58

- Biochemical tests identify bacterial species by differentiating them based on biochemical activities. - Grow bacteria on different growth media containing specific components. - tests for specific enzymes/ biochemical pathways. - Can be detected by simple tests, e.g. pH colour change. - Commonly performed using commercial kits such a API strips. - Can show if they can use different substrates for growth, e.g. metabolize sugar aerobic or anaerobic

Answer 59

MIC: The [lowest] of antibiotic allowing NO growth of the bacteria. Compare MIC to the break point to determine if the bacteria is susceptible or resistant. Breakpoint: the highest [plasma] of the drug that can safely be achieved in a patient to define susceptibility to an organism. It is a cut-off value that defines isolates as susceptible or resistant in clinical practice. (If the MIC value > than breakpoint then the pathogen is resistant)

Answer 60

Oxidase Test: for cytochrome oxidase (involved in respiratory chain)

Answer 61

Citrate Test: ability to use citrate as carbon source Agar contains citrate & pH indicator (bromothymol blue) green: negative (remains neutral) blue: positive (alkaline)

Answer 62

Urease Test Identifies urease, an enzyme that hydrolyses urea to ammonia & CO2 Medium contains pH indicator (phenol red) & urea After incubation: positive: PINK medium alkaline- negative: YELLOW medium acidifies

Answer 63

Indole Test: Presence of the enzyme tryptophanase

Answer 64

Pros: - Regarded as the ‘gold standard’ - Live culturable cells - Ensures you are looking at a single species - Provides accurate sensitivity testing. - Cheap Cons: - Lengthy- takes 48 hours or longer - Errors can happen & the wrong organism can be cultured. - Need for antimicrobial to be started before MC&S has been completed. - Rare or unusual or non-culturable organisms will be missed. - Need to isolate a single/ pure colony prior to stating sensitivity or biochemical testing.

Answer 65

- Polymerase chain reaction (PCR), uses repeated cycles of heating & cooling to copy a specific piece of DNA. - Pathogen DNA (or RNA Virus) isolated from the sample is the template - Primers: Short pieces of DNA, are designed with a sequence specific for the pathogen. - 2 primers used – one for each end of the gene sequence= primer pair)

Answer 66

Template- Pathogen DNA Primer pair. Specific to pathogen Nucleotides-----A T C G DNA polymerase. Enzyme which Extends the primers Buffer- to make sure it works

Answer 67

1. Denature. Template DNA unzips, and separates 2. Anneal. Specific DNA primers attach onto target DNA 3. Extend. DNA polymerase adds single nucleotides to the primer to make an identical piece of DNA Cycle repeats 30+ times, each cycle uses the newly made product as a template

Answer 68

Quantitative PCR: Fluorescent reporter added to PCR reaction for measuring DNA generation during the qPCR assay. Fluorescence signal accumulates as the PCR proceeds - Can determine if sample is positive - Can quantify amount of PCR product present at a given cycle. - Can quantify amount of pathogen in the sample – Particularly useful for determining viral load. - Sometimes called Real Time PCR RT-PCR

Answer 69

Rapid screening for antibiotic resistance genes qPCR can be used for the detection of SOME common antibiotic resistance genes: E.g. mecA - MRSA NDM1 (Plasmid encoded ESBL-carbapenem resistance in bacteria of the family Enterobacteriaceae CRE) VanA gene –resistance to Vancomycin/ teicoplanin. Rapid tests for resistance in TB The presence of the gene doesn’t tell if it’s expressed, or if the bacteria is resistant. MC&S is the only method which definitively tells you this. Sequencing PCR product to find point mutations possible Not usually feasible in a diagnostic lab.

Answer 70

qPCR multiplex assays: - Can amplify several DNA fragments using different fluorescent reporters. - Can detect several pathogens in a sample that are implicated in particular infections: E.g. bloodstream infections, community-acquired pneumonia, meningitis, sexually transmitted diseases urinary tract infections

Answer 71

Positives: - Process is quicker than culture methods - Can extract & test DNA directly from your sample. - Detect viruses ‘& non-culturable’ pathogens - Detect difficult to culture pathogens (e.g. TB) -Can quantify viral load - Detects multiple pathogens (multiplex) - Can detect some resistance genes - Easy to adapt to novel pathogens (design specific primers) Negatives: -Expensive - Requires a dedicated lab. - Need Good QA, QC & controls to prevent false negatives/ positives - Can only detect something if using appropriate primers. - Need to have enough genetic material in initial sample - Presence of resistance gene doesn’t mean pathogen is resistant.

Answer 72

- Use universal primers which are less specific. - Example: DNA from most species of bacteria will produce a ‘PCR product’ with primers to the 16s rRNA Gene. - The precise sequence of this gene differs in different species. - Can sequence PCR product & identify species using databases. - Only works on a pure culture. - 16S gene highly conserved but contains hypervariable regions - Hypervariable regions: wide variation in sequences between species - Organisms grouped on similar hypervariable regions

Answer 73

- Sometimes called “massively parallel sequencing” - A collection of technologies which can rapidly sequence the whole genome broken down into short reads with high throughput. - Doesn’t need specific primers - (use an adapter) - Rapid sequencing of whole genomes. - Identification of mutations & epigenetics

Answer 74

- Mass spectrometry: Chemical compounds are ionized into charged molecules & the ratio of their mass to charge (m/z) is measured. - MALDI: Matrix assisted laser desorption ionization. Method which allows the mass spectrum of large molecules to be analysed e.g. proteins

Answer 75

MALDI-TOF MS methodology: - Sample is co-crystallized with a matrix on the sample target. - Ionized by the MALDI ion source (e.g. ultraviolet laser). - The charged molecules, (including microbial peptides/proteins), are accelerated by the electric field into the Time of flight (TOF) analyser. - Ions separated by TOF based on mass to charge (m/z). - Detected & a mass spectra produced. - This ‘fingerprint’ is compared with a database of known organisms

Answer 76

- Can only identify bacteria from a pure sample - (Not a mixed culture) - Sample can be a single colony from a plate. Direct transfer of colony to the matrix - Some organisms/ sample types might need an extraction method - Can be used to identify blood stream infection (BSI) directly from blood cultures - Rapid identification (30 mins compared to 18-24 more hours) - Can be used with samples isolated directly from CNS-----Eg identification of causative agent of meningitis - Can be used on clinical urine samples (high number of bacteria present.)

Answer 77

- Diagnostically possible to identify some types of resistance. - E.g. some ESBL producing bacteria, - But may need to grow bacteria in the presence of antibiotic & then detect breakdown products - Commercially available methods for detecting some AMR in BSI.

Answer 78

Pros: - Automated systems can give very rapid results (eg 30 mins) - Accuracy- Can identify bacteria to strain level (Particularly Gram-negative bacteria) - High throughput. - Can be used directly on positive blood cultures - Technician time reduced (less human error/ cost) - Technician training required is low - Cost per sample is cheaper compared to PCR Cons: Can’t identify bacteria from a mixed population. E.g. Polymicrobial BSI problematic - Presence of mixed population may lead to false identification - Accuracy - Based on quality of database - Need reference spectra to allow identification - Different preparation methods for different bacteria - Hard to distinguish some types of bacteria: Closely related species Gram positive bacteria (similarity of cell wall) Encapsulated species– eg Klebsiellea - Some samples need complex preparation e.g. Mycobacteria, mold, fungi. - Expensive

Answer 79

- Used to detect viral & bacterial antigens & antibodies, to help diagnose diseases & check immune status. - Huge range of methods: ELISA, chemiluminescence, agglutination, direct & indirect immunofluorescence - Enzyme Linked immunosorbent assay (ELISA): 1. Antibodies coat base of plate 2. Antibodies capture specific target antigen from sample 3. A 2nd enzyme-labelled antibody forms a ‘sandwich.’ 4. The enzyme reacts with a substrate to create a detectable signal - Can detect: antibodies, antigens, proteins, Glycoproteins Quantitative or qualitative

Answer 80

- Detect target in a liquid sample. - Can test for specific antigens e.g. Covid19 - Hormones (pregnancy) etc.

Answer 81

Pros: - Range of different tests available - Different applications - Can be quick, easy & cheap - May be used at Point of care (POC) - More complex tests can be quantitative (can reach max limit quickly) - Some test have high specificity - Usually qualitative Cons: - Detection of antibodies in a patient’s sera is retrospective- 2-4 weeks post infection - More sophisticated tests require specialist reagents, equipment and technical knowledge - Can only look for specific known antigen- not always known - Individual’s serum reaction is unpredictable - Lateral flow test - Less sensitive than PCR

Answer 82

Gram + Thick peptidoglycan cell wall retains crystal violet stain (blue/purple) e.g. Staphylococcus aureus Gram – (pink/red) Thin peptidoglycan cell wall does not retain crystal violet stain, does counterstain (pink/red) e.g. E. coli “Atypical” aren’t detected on Gram stain. E.g. M. tuberculosis

Answer 83

Beta-lactamase sensitive ("penicillins"): Act mainly on G+ (some activity against G-ve) e.g. benzylpenicillin used ASAP IV in suspected meningococcal disease in the community

Answer 84

Beta-lactamase resistant: e.g. flucloxacillin: narrow spectrum beta-lactam antibiotic used against staphylococcal infections producing b-lactamases. Acid stable: can be given orally.

Answer 85

Anti-pseudomonal: Broad spectrum agents used to treat pseudomonas infection e.g. Tazocin: Piperacillin + taxobactam (b-lactamase inhibitor), given iv

Answer 86

Broad spectrum: e.g. amoxicillin active against G+ve & most G-ve. inactivated by b-lactamases; b-lactamase inhibitors sometimes added e.g. co-amoxiclav; Amoxicillin + clavulanic acid (b-lactamase inhibitor)

Answer 87

Broad spectrum antibacterials act against a wide range of bacteria, usually including both Gram positive and Gram negative bacteria. E.g. Tazocin. Narrow spectrum antibacterials have a narrower range of activity (usually either Gram + or Gram -). e.g. Metronidazole

Answer 88

Antimicrobial stewardship – preserving their effectiveness Broad spectrum antibiotics are associated with selection of antimicrobial resistant microbes. Avoiding patient harm Using indiscriminate broad-spectrum antimicrobials can increase the risk of collateral damage to the patient’s gut microbiota & increase the risk of subsequent opportunistic infection (for example C. difficile) & inflammatory bowel disease.

Answer 89

The two methods to select an antibacterial: Directed therapy: Based on knowing what the pathogen is +/- sensitivity to antibacterial Empirical therapy: Based on probability

Answer 90

Traditionally MC&S (microscopy, culture and sensitivity) Stepwise provision of information: 1. Culture positive (something is growing) 2. Identification (we know what it is) 3. Sensitivities (we know what to treat it with)

Answer 91

Pros: 1. know infection cause 2. know resistance 3. more successful treatment 4. Targeted antibiotic therapy 5. Can often use narrower spectrum agent 6. Less chance of nosocomial infection Cons: 1. might still be wrong: could be contaminant or colonisation 2. There might be mixed cause, all organisms might not have grown 3. Some tests take time

Answer 92

Pros: 1. start therapy quickly 2. Should be effective in most cases 3. Evidence-based Cons: 1. sometimes wrong 2. Causative organism might be resistant 3. Tends to be broader spectrum: more risk of side effects & nosocomial infections & generating resistance 4. Giving antibiotics may reduce probability of a successful culture (take samples first)

Answer 93

Eradication difficult: 1. endocardium poorly vascularised 2. bacteria buried among platelets and fibrin in “vegetations,” make access harder for antibiotics 3. slow-growing pathogens display antimicrobial tolerance Empirical treatment should be started promptly Draw 3 sets of blood cultures at 30-minute intervals before starting antibiotics Once the pathogen is identified, change to directed therapy within 24-48h

Answer 94

1. First-line for uncomplicated urinary tract infections 2. Renal impairment: antibacterial efficacy depends on renal secretion into the urinary tract; risk of peripheral neuropathy increased in renal impairment. 3. Wide range of side effects: in particular need for vigilance for pulmonary & hepatic adverse drug reactions. 4. Monitoring: On long-term therapy, monitor liver function & monitor for pulmonary symptoms, especially in the elderly (discontinue if deterioration). 5. Trimethoprim may be used instead if renal function is impaired.

Answer 95

1. Nitrofurantoin & renal impairment: Risk of peripheral neuropathy increased in renal impairment; antibacterial efficacy depends on renal secretion of the drug into urinary tract. 2. Trimethoprim: resist is an issue, only use when local data suggests resist is not issue 3. Gentamicin: im/iv, ototoxic, nephrotoxic, needs monitoring 4. Ciprofloxacin: Rare disabling adverse rxn profile (convulsions & tendon rupture) – risks>benefit, only for severe infections. 5. Broad spectrum antibiotics: Use unwarranted to cover E.coli without extended spectrum beta lactamases

Answer 96

Directed therapy gives info about resistance in individual patient’s infection. Empirical therapy gives info about risk of resistance e.g. known patterns of resistance in population, previous antibacterial history, previous culture results

Answer 97

1. male 2. pregnant 3. catheterised 4. immunosuppressed 5. Upper UT symptoms 6. Recurrent

Answer 98

1. Severity of symptoms 2. Risk of complications 3. Complicated” UTI: 4. Antibacterial effectiveness vs pathogen 5. RESISTANCE 6. Route/ oral bioavailability 7. Side effects (ciprofloxacin) 8. Tissue penetration/ excretion route 9. Narrow/ broad spectrum 10. Renal function (e.g. nitrofurantoin

Answer 99

1. Acute illness (21 days) with cough, & 1 or more of (fever, sputum production, breathlessness, wheeze, chest pain) 2. no alternative explanation (e.g. sinusitis, asthma). 3. LRTI includes acute bronchitis, pneumonia, other infections in the LRT 4. Pneumonia: infection of the alveoli (air sacs) of the lungs

Answer 100

Acute bronchitis: 1. History: Cough +/- sputum, wheeze, shortness of breath (SOB), possible coughing pain 2. Chest exam: Mildly ill, Wheeze 3. General Exam: May have systemic features & fever 4. Chest X-ray: Normal

Answer 101

Pneumonia: 1. History: Cough +/- sputum, shortness of breath, pleural pain, fever, aches & pains 2. Chest Exam: Moderate - severely ill, Focal chest signs e.g. coarse crackles, dull to percussion 3. General Exam: Fever ≥38°, raised heart rate, respiratory rate, may be confused. 4. Chest X-ray: Chest Xray changes (new infiltrate confirms pneumonia)

Answer 102

Antibiotic used dependant on: 1. Co-morbidity 2. Risk of adverse effects from broad spectrum antibiotics 3. Risk of complications 4. Recent antibiotic use 5. Resistance 6. Recent microbiology results 7. Local surveillance data 8. Causes

Answer 103

Antibiotic should be given within 4 hours of pneumonia diagnosis (CAP or HAP), as within 1 hour if at risk of sepsis.

Answer 104

Community-acquired (CAP) Common causes include: 1. Gram + S. pneumonia, S. aureus 2. Gram - K. pneumoniae, H. influenzae 3. Atypical L. pneumophila, M. pneumoniae, C. pneumoniae, C. psittaci 4. Viral

Answer 105

Hospital-acquired (HAP) Onset ≥48 hours after hospital admission: 1. As CAP but also 2. More resistance e.g. MRSA, multi-drug resistant Pseudomonas aeruginosa, ESBLs 3. Wider range of pathogens e.g. Escherichia coli, Klebsiella pneumoniae, Acinetobacter species 4. Different pneumonia sources have different profiles, resist, patient factor & outcomes. 5. If in hospital for >2 days, treated as HAP, leading to different treatment.

Answer 106

Risk of mortality in community-acquired pneumonia: CRB-65 score. <1% Low, 1-10% Moderate, >10% High. Determined with: 1. Confusion: 2. Respiratory rate raised, ≥30 breaths per minute 3. Blood pressure low, diastolic ≤60 mmHg or systolic ≤90 mmHg). 4. ≥65 years in age 5. Can add Urea for CURB-65 in hospital setting

Answer 107

Unusual presentations can suggest atypical Legionella pneumonia: 1. dry cough 2. no fever 3. headache 4. confusion 5. diarrhoea 6. hyponatraemia

Answer 108

Unusual presentations can suggest atypical: Mycoplasma pneumonia: 1. upper respiratory involvement 2. skin changes 3. encephalitis 3. uveitis, myocarditis 4. haemolytic anaemia

Answer 109

1. For CAP, patients given antimicrobials based upon severity, determined by CRB65 score. 2. Guides treatment, including allergies. 3. This minimises downsides of broad-spectrum antibiotics 4. Assists in slowing the adaption of antibiotic resistance.

Answer 110

CAP 1. Low severity: mostly aiming to cover S. pneumoniae 2. Amoxicillin: good vs. S. pneumoniae, low adverse effects & resist rates. 3.Doxycycline, clarithromycin, erythromycin: good vs. S. pneumoniae, & broad spectrum of activity 4. Increasing severity – covering broader range of pathogens, particularly atypical HAP: More resist problems, wider pathogen range needed, more likely use of broad-spectrum antibiotics

Answer 111

Patient factors influencing choice of antibacterial 1. Allergies 2. Renal and hepatic impairment 3. Drug interactions 4. Other contraindications e.g. pregnancy 5. Immunosuppression 6. Other co-morbidities 7. Risk factors for severity 8. Risk of resistance

Answer 112

Immunosuppression: 1. Increases risk of severe infection and complications 2. Changes profile of likely pathogens 3. CAP: atypical/P. aeruginosa more likely 4. Need to consider broader range of pathogens, including fungal pneumonia 5. Opportunistic infections: occur more frequently & more severe in people with weakened immune systems 6. Asplenia: absence of spleen. 7. At risk from encapsulated bacteria, including S. pneumoniae, H. influenzae

Answer 113

Renal impairment: 1. Increases risk of infection 2. Is the drug effective in renal impairment 3. Will it affect excretion of the antibiotic? 4. Is a dose reduction needed in renal impairment e.g. amoxicillin 5. No adjustment needed for metronidazole 6. Is the antibiotic nephrotoxic?

Answer 114

Hepatic Impairment 1. Increases risk of infection 2. Can affect absorption, distribution & clearance of drug - e.g. metronidazole metabolised in liver - reduce dose in severe hepatic dysfunction 3. Hepatic reserve limited – avoid or caution with drugs which can be hepatotoxic e.g. flucloxacillin

Answer 115

Co-morbidities: 1. Immunosuppression 2. Renal and hepatic impairment 3. Others e.g. epilepsy: Quinolones (e.g. ciprofloxacin) LOWER seizure threshold (interacts with phenytoin, antiepileptic drug)

Answer 116

Ciprofloxacin (P450 enzyme inhibitor): Decreased metabolism (increased levels) for many drugs metabolised in liver by cytochrome P450 enzymes e.g. warfarin Rifampicin (potent INDUCER of P450 enzymes): Increased metabolism (decreased levels) of other drugs including warfarin (may need 2-5 fold dose increase), progesterones (COC & POP hormonal contraceptives) Macrolides, especially erythromycin (P450 inhibitor - isoform CYP 3A4): Decreased metabolism (increased) levels of other drugs. E.g. warfarin Risk of myopathy with statins – tend to stop statins for duration of course & 2 days after

Answer 117

Alcohol + Metronidazole  ‘disulfiram-type’ reaction: vomiting, abdominal pain, palpitations, flushing. Avoid -OH for 48 hours Linezolid – used for G+ bacteria including MRSA – monoamine oxidase inhibitor (MAOI) – avoid fermented drinks e.g. beer

Answer 118

1. Avoids cannula-related infections 2. Better patient experience 3. Easier in community 4. Cheaper 5. Reduces broad-spectrum antimicrobial exposure – better antimicrobial stewardship

Answer 119

1. Usually given IV to treat Gram + infections E.g. skin & soft tissue infections, bone & joint infections, CAP/HAP/VAP 2. Orally to treat C. difficile infections 3. Diarrhoea & colitis due to bacterial infection with C. difficile 4. Almost always occurs during or after antibacterial use – stop existing antibiotics if possible 5. Other risk factors: older age, in hospital, proton pump inhibitor (e.g. omeprazole), previous C. diff, immunosuppression

Answer 120

1. Used for severe life-threatening infections (e.g. sepsis, meningitis) 2. Possibly needed due to oral bioavailability of drug or patients who are unable to take or absorb oral drugs (e.g. vomiting) 3. Prolonged iv for ‘deep-seated’ infections (e.g. bones & joints, infective endocarditis)

Answer 121

1. Bacterial meningitis: bacterial infection of the protective membranes that surround the brain & spinal cord (meninges). 2. Commonest causes S. pneumoniae & N. meningitidis. Older adults & vulnerable groups, wider aetiology including L. monocytogenes. 3. In hospital, first line empirical therapy is iv ceftriaxone (+ iv amoxicillin if risk factors for Listeria) 4. Beta-lactams typically poor penetration of cerebrospinal fluid, but: 5. High level of activity against relevant pathogens 6. Relatively low toxicity – can use high dose 7. Penetration improved with N. meningitidis inflammation

Answer 122

Meningitis = Inflammation of protective membranes surrounding the brain & spinal cord (meninges). Causes include bacterial, viral & fungal infection, & non-infective. Both following must be reported to Health Protection Team. Bacterial meningitis: bacterial infection of the meninges. E.g. S. pneumoniae Meningococcal disease: infections with N. Meningitidis “meningococcus”

Answer 123

Strongly suspect bacterial meningitis with the red flag combination: 1. fever 2. headache 3. neck stiffness 4. altered level of consciousness or cognition (including confusion or delirium) Can be present with none of the symptoms present

Answer 124

Symptoms can include: 1. non-blanching rash 2. looking unwell 3. fever 4. lethargic or unusual behaviour 5. confusion 6. altered consciousness 7. cold hands & feet 8. skin pale/mottled/cyanosed (bluish) skin 9. abdominal pain 10. diarrhoea 11. leg pain.

Answer 125

1. If cause known: iv amoxicillin 2. Cause unknown: Ceftriaxone, if immunosuppressed, pregnant or >60 add iv amoxicillin

Answer 126

1. How does the patient feel? 2. Physical symptoms relevant to the infection 3. Physiological parameters: e.g. blood pressure, heart rate 4. Possible complications: e.g. renal function deteriorating? 5. Relevant blood tests: e.g. White Cell Count (WCC) & C-reactive protein (CRP) decreasing

Answer 127

1. Monitor for side effects relevant to antibiotic. 2. Therapeutic levels to ensure antibacterial is effective while preventing toxicity. 3. Monitoring common antibacterials: Vancomycin, Gentamicin, Teicoplanin 4. These Drugs can only be used in settings with facilities to monitor levels & interpret the results.

Answer 128

1. Nephrotoxicity: usually reversible, drug accumulates in proximal tubule 2. Ototoxicity: Irreversible, drug accumulates in fluid-filled inner ear & damages hair cells 3. Reduce risk with therapeutic drug monitoring. 4. Single daily doses least toxic.

Answer 129

1. Antibiotic-associated diarrhoea relevant for all antibiotics, particularly broader-spectrum. 2. Includes C. difficile associated diarrhoea 3. Can be life-threatening 4. Watery diarrhoea, fever, increase white cell count 5. Especially a problem with aminopenicillins (e.g., amoxicillin), cephalosporins (e.g., ceftriaxone), clindamycin 6. Candidal infections

Answer 130

1. Relevant for all antibiotics. Symptoms: fever, hives, rash. 2. Anaphylaxis mediated by IgE – requires prior exposure. 3. Important for penicillin e.g. amoxicillin 4. Allergic to one, allergic to all penicillin, may be allergic to other beta-lactams e.g. cephalosporins. 5. Patients with anaphylaxis history, hives or rash immediately after penicillin administration at risk of immediate hypersensitivity: should not receive any penicillin or other beta-lactams.

Answer 131

1. Reaction to infusion: usually first or fast infusion of vancomycin 2. Itchy red rash, flushed 3. Can cause low blood pressure, angioedema 4. Driven by histamine 5. Slowly give Vancomycin to reduce risk

Answer 132

Many antibacterial are nephrotoxic, neurotoxic, or both 1. Gentamicin: Nephrotoxic & ototoxic 2. Vancomycin: Nephrotoxic (and, rarely, ototoxic) 3. Penicillins: Neurotoxicity e.g. seizures (renal impairment increases risk) 4. Chloramphenicol: Optic neuritis 5. Polymixins – nephrotoxicity & neurotoxicity limit use, but with increasing AMR these are used again for serious infections due to selected aerobic Gram- bacteria in patients with limited treatment options.

Answer 133

1. Neutropoenia (low levels of neutrophils) & agranulocytosis (dangerously low WBC e.g. neutrophils). 2. Can be caused by a range of antibiotics including vancomycin, trimethoprim. 3. e.g. Chloramphenicol can cause aplastic anaemia: bone marrow stem cells die. Rare but can be fatal.

Answer 134

1. Cardiac arrhythmias e.g. erythromycin - QT interval prolongation 2. Cholestatic jaundice – erythromycin 3. Hepatotoxicity – e.g. rifampicin, tetracyclines, co-trimoxazole 4. Pregnancy/ breastfeeding/infants e.g. chloramphenicol – Grey baby syndrome (abdominal distention, haemodynamic collapse, grey skin) 5. Some relevant with interactions e.g. erythromycin + statins – risk of myopathy (hold statins).

Answer 135

Safety netting - Advising people what to do if: 1. their condition deteriorates 2. does not improve within a certain time, 3. if they develop adverse effects caused by the treatment.

Answer 136

Cease antimicrobial prescription if no evidence of infection Amend antimicrobials – ideal to narrower spectrum agent – ensure treatment is effective & proportionate. Refer to non-ward based antimicrobial services to reduce risk of acquisition of HAI Extend antimicrobial prescription & document review date or stop date. Switch antimicrobials from IV to PO according to national IV to PO switch (IVOS) criteria.

Answer 137

Close contacts: 1. Some infections require identification & treating of close contacts e.g. meningitis 2. Seek advice form local health protection team to identify & manage contacts 3. Antibiotic prophylaxis (antibiotics preventing disease) should be given asap, ideally within 24 hours from case diagnosis. Close contact assessment for meningitis/meningococcal disease usually based on: 4. Prolonged close contact for 7 days e.g. household 5. Transient close contact if expose to large particle respiratory sections e.g. resuscitation

Answer 138

Risk factors Invasive fungal infections: 1. Main: immunocompromised patients 2. Haematological malignancies 3. Cancer – chemotherapy 4. Organ or bone marrow transplant 5. AIDS/HIV 6. Broad spectrum antibiotics (loss of gut flora) 7. Indwelling catheter, in particular central IV line 8. Respiratory diseases, e.g. cystic fibrosis, Covid-19

Answer 139

Most common: Aspergillus fumigatus Filamentous mold – form mycelium Saprophytic – live on organic matter 1. Spores are breathed in 2. If healthy, cilia remove them 3. If immunocompromised they invade & neutrophils are recruited 4. Can cause lung damage 5. Can cause Neutropenia, excessive growth in the lung which can spread

Answer 140

Aspergillosis: Invasive aspergillosis (including disseminated) – typically of lung origin ABPA (allergic bronchopulmonary aspergillosis) Aspergilloma (fungal ball), difficult to remove Growth of fungal ball in pre-existing cavity e.g. patient with previous TB

Answer 141

1. Dimorphic yeast 2. Yeast when harmless in gut 3. Mycelium when invasive within the lungs & form germ tubes

Answer 142

1. Damage possibly from surgery, 2. Migrate out of gut, move between cells & form mycelium 3. Form a central line, may develop into a biofilm 4. Then cause disease, e.g. Candidia in the blood, or endocarditis

Answer 143

1. Highly resistant – some isolates are pan-resistant (resist all antifungal) 2. Several outbreaks worldwide, including in some UK hospitals, usually ICU 3. Thermotolerant – can grow up to 42oC

Answer 144

1. Lack of routing diagnostic testing for fungal diseases. This could lead to, for instance inappropriate use of broad-spectrum antibacterials 2. Clinical diagnosis: medical history, symptoms, and risk factors 3. Cultures of blood, respiratory (sputum, BAL), or biopsy samples slow and not very sensitive; some species unculturable on routine culture media 4. Direct microscopy of clinical samples: poor sensitivity, requires expertise in fungal identification 5. Imaging: X-ray/CT

Answer 145

1. Serology-based: specific antigen detection 2. A component of the cell wall is one three beta glucan 3. There is a test to detect this specific sugar. All fungi have this beta glucan. 4. There are two of this beta glucan, one is detectable, but tests are often false. 5. Aspergiullus: Galactomannan can be used 6. Can use PCR to detect nucleic acids

Answer 146

Echinocandins inhibits cell wall synthesis Caspofungin inhibits B-glucan synthase B sits in membrane links layer of B glucan layer. Inhibits making of 1,3 B-glucan, weakening the cell wall Azoles inhibit step in synthesis of ergosterol Causes accumulation of molecules, membrane become leaky Cell stops growing Amphotericin B directly binds to ergosterol & creates pores & causes leaks in the membrane

Answer 147

1. Decreased ergosterol production through inhibition of fungal cytochrome p450 enzymes. 2. Most are fungistatic 3. Orally active 4. Side effects: Liver toxicity, QT prolongation 5. Interactions Through cytochrome p450 enzymes – contraindicated with drugs that induce (eg rifamycin) or are metabolised by cyt P450 enzymes (some statins)

Answer 148

Fluconazole: Tablet, 24hr half life, weak CYP 3A4 inhibition but excellent CSF penetration. Itraconazole: Solution, 16-28hr half life, Strong CYP 3A4 inhibition, poor CSF penetration Voriconazole: Tablet, 6hr half life, excellent CSF penetration, moderate-strong CYP 3A4 inhibtion Each cause Nausea & Vomiting

Answer 149

1. Azoles used as fungicides in agriculture, leads to resistance 2. Efflux transporters 3. Changing target of medication 4. Expressing more of target

Answer 150

1. Interacts directly with ergosterol, creating pores in the membrane 2. Broad spectrum - active against most fungi 3. Not orally absorbed 4. For severe systemic infections only 5. Significant side effects – toxicity is limiting factor as may bind to cholesterol & cause our cells to become leaky 6. May be combined with flucytosine

Answer 151

1. Amphotericin B (Fungizone) has renal toxicity, damages tubules in kidney. 2. Toxicity reduced with lipid formulation e.g. Ambisome - liposomal, Abelcet - lipid complex 3. Now usually used first line over amphotericin B, lower toxicity 4. All formulations require test dose (1 mg) first, & observe over 30 min

Answer 152

1. Inhibit 1,3-β-glucan synthase 2. Very poor oral bioavailability; IV only 3. Toxicity low as we don’t have targets for drug, & minimal drug interactions

Answer 153

1. Ibrexafungerp (brexafemme; GSK) 2. Inhibits 1,3-β- glucan synthase – but different MoA from echinocandins 3. FDA approval in 2021 for vaginal candidiasis. In clinical trials for IV 4. Active against Candida; first orally available inhibitor of 1,3-β- glucan synthase

Answer 154

1. Olorofim (F2G) 2. Inhibits pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase 3. Phase 2. FDA: breakthrough therapy & orphan drug status 4. Broad spectrum & active against highly resistant molds

Answer 155

1. Fosmanogepix (Pfizer) 2. Inhibits GPI anchor post translational protein modification 3. Phase 2. FDA: fast track & orphan drug status 4. Broad spectrum and active against highly resistant fungi

Answer 156

1. Improving Patient Outcomes: 2. Reducing Antibiotic Resistance 3. Minimizing Adverse Effects: 4. Decreasing Healthcare Costs 5. Enhancing Infection Control

Answer 157

1. Reduce incidence of adverse antibiotic associated events - C. difficile infection (CDI) - Antibiotic associated diarrhoea (AAD) - Allergic reactions & other toxic effects 2. Reduce resistance: Reserve some antibiotics to preserve effectiveness? 3. Pharmacoeconomics: Practice cost-effective prescribing

Answer 158

1. C. difficile causes disease when the normal bacteria in the gut are disadvantaged, due to antibiotics. 2. Allows C. difficile to grow & allows toxins some strains produce to reach levels where it attacks the intestines & cause mild to severe diarrhoea. 3. C. difficile can lead to more serious infections of the intestines with severe inflammation of the bowel (P. colitis).

Answer 159

S Suspect infective case where there no clear alternative causes I Isolate patient & consult the infection control team G Gloves & aprons H Hand washing T Test the stool for toxin

Answer 160

1. Faecal Microbiota Transplant recommended to treat recurrent C.difficile in adults who have had 2 or more previous confirmed episodes. 2. Aims to restore a healthy gut microbiome. 3. Involves transferring intestinal bacteria & other microorganisms from healthy donor faeces into the gut of the recipient.

Answer 161

1. Adsorption: Virus attaches to host cell, binding spikes to cell receptors 2. Penetration: Virus engulfed by vesicle by endocytosis 3. Uncoating: Envelope is removed, RNA is freed into cytoplasm 4. Synthesis: Unver viral gene control, cell synthesizes base components of new viruses 5. Assembly: Viral spikes insert into cell membrane for viral envelope, nucleocapsid formed from RNA & capsomers 6. Release: Enveloped viruses bud off membrane, carrying away envelop with spikes. Complete virus is ready to infect another cell.

Answer 162

1. Viruses are not ‘alive’, so they can not be ‘killed’. 2. Viruses utilise many host proteins for their replication. 3. Viruses have simple structures, often few druggable targets exist. 4. Some viruses mutate rapidly and so acquire resistance to drugs.

Answer 163

Herpes virus Features: 1. Encode many enzymes – many drug targets 2. Establish latent infections 3. Lifelong persistence 4. Significant cause of death in immunocompromised hosts 5. Some can cause cancers 6. Comprised of three sub-families: alpha, Beta, gamma

Answer 164

All herpesviruses have identical morphology & are indistinguishable from each other under electron microscopy. 1. Alpha herpesviruses (main herpes): 2. Beta herpesviruses: 3. Gamma herpesviruses:

Answer 165

Alpha herpesviruses (main herpes): 1. Herpes simplex virus type 1 (HSV-1) 2. Herpes simplex virus type 2 (HSV-2) 3. Varicella-zoster virus (VZV)

Answer 166

Beta herpesviruses: 1. Cytomegalovirus (CMV) 2. Human herpesvirus 6 (HHV-6) 3. Human herpesvirus 7 (HHV-7)

Answer 167

Gamma herpesviruses: 1. Epstein-Barr virus (EBV) 2. Human herpesvirus 8 (HHV-8)

Answer 168

1. HSV-1: oropharyngeal sores (children) 2. HSV-2: genitalia (young adults) 3. Man is the only natural host for alpha herpesvirus. 4. Virulence factors: gC binds complement C3b (innate immune system) gE is an Fc receptor for IgG, blocking disease specific memory antibodies (adaptive immune system

Answer 169

HSV-1 (oral Herpes) 1. Commonly acquired by children 2. Most adults are seropositive 3. Only a small proportion have recrudescence

Answer 170

HSV-2 (genital Herpes) 1. Commonly acquired by young adults 2. STD 3. Fetal/newborn transmission 4. Increased risk for HIV infection

Answer 171

1. HSV spreads by contact, virus is shed in saliva, tears, genital & other secretions. 2. Most common infection results from kiss given from a person shedding the virus 3. 2 peaks of incidence. 1st 0-5yrs (HSV-1). 2nd late teens (HSV-2), when sexual activity commences.

Answer 172

Replication of HSV-2: 1. After attachment, host cell membrane fuses with viral envelope, permitting entry of nucleocapsid to cytoplasm. 2. Viral capsid is uncoated & viral genome DNA enters the cell’s nucleus 3. New viral DNA is synthesised in the nucleus 4. Transcription produces mRNA that are translated on the cytoplasmic ribosomes into capsid & spike proteins 5. Capsid proteins enter nucleus & combine with viral genomes to form new nucleoplasts 6. Viruses bud through nuclear membrane, receives spiks at Golgi compartment. Exocytosis releases the new virions.

Answer 173

1. Structural analog Guanosine 2. Inhibition of viral synthesis of DNA 3. Topical, oral, & IV formulations 4. Low oral bioavailability 5. Acts on DNA replication, causing broad spectrum: Herpes simplex 1 & 2, varicella-zoster virus (alpha sub-family), possibly Epstein-Barr Virus (gamma sub-family).

Answer 174

1. Acyclovir is a pro-drug. Must be phosphorylated to acyclovir tri-phosphate to be active, as nucleotide triphosphate is used in DNA synthesis. 2. Is incorporated into replicating viral DNA strand & blocks further replication. 3. Preferential incorporation by viral DNA polymerase 4. Viral DNA polymerase ‘prefers’ Acyclovir-TP over the natural GTP. When Acyclovir is introduced there is no new 3’-OH, so replication cannot continue. 5. Normally DNA polymerase introduces new nucleotide resulting in new 3’-OH so replication continues. 6. 1st phosphorylation step carried out by viral thymidine kinase, meaning this process only occurs in infected cells, as human kinase is more specific & won’t act upon the acyclovir. 7. Subsequent phosphorylation reactions are carried out by host kinases.

Answer 175

Valacyclovir: 1. Ester exists to protect hydroxyl, allowing drug to be more taken up when administered orally. 2. L-valyl ester prodrug of acyclovir, rapidly & almost completely converted to acyclovir 3. Available orally only 4. Spectrum: similar to acyclovir (alpha sub-family) 5. Adverse effects: headache, nausea, weakness, dizziness, confusion

Answer 176

Famciclovir 1. Cyclic guanine analogue 2. Converted to pencyclovir in the liver & intestines 3. Available orally only 4. Spectrum: HSV-1 and-2, VZV, to a lesser extent, EBV, 5. Adverse effects: headache, GI

Answer 177

Varicella-Zoster virus: 1. Belong to alpha herpesvirus subfamily 2. Double stranded DNA 3. Enveloped virus 4. One antigenic serotype only

Answer 178

1. Virus enters via respiratory tract & spreads to the lymphoid system. 2. After incubation, virus arrives at the skin. 3. Following primary infection, virus remains latent in cerebral or posterior root ganglia. May cause a single recurrent infection occurs after several decades. 4. Virus reactivates in ganglion & tracks down the sensory nerve to the area of the skin supplied by the nerve, producing a varicella form rash in the distribution of a dermatome.

Answer 179

1. Primary infection results in varicella (chickenpox) 2. Presents fever and a widespread vesicular rash, possibly fever 3. Typically self-limiting, complications are rare but occurs more frequently and with greater severity in adults and immunocompromised patients. 4. Most common complication is secondary bacterial infection of the vesicles. 5. Severe complications which may be life threatening include viral pneumonia, encephalitis, and hemorrhagic chickenpox.

Answer 180

1. Herpes Zoster mainly affects a single dermatome of the skin. 2. Majority of patients >50 yrs old 3. Latent virus reactivates in sensory ganglion & tracks down the sensory nerve to the appropriate segment. 4. Characteristic eruption of vesicles in the dermatome, often with pain 5. Herpes zoster can affect the eyes & face 6. Greater problem in immunocompromised patients in whom reactivation occurs earlier in life & multiple attacks occur as well as complications.

Answer 181

1. Uncomplicated is self-limited disease & requires no specific treatment. 2. Acyclovir shown to accelerate resolution of the disease 3. Acyclovir given promptly to immunocompromised & normal individuals with serious complications e.g. pneumonia & encephalitis.

Answer 182

HIV structure: 1. Retrovirus use single stranded RNA to hold genetic info. 2. During replication cycle, the virus must convert the RNA into DNA, a rare exception. 3. Contains: 2 copies of RNA, Enzymes (Reverse Transcriptase, Integrase, Protease) 4. Two major envelope proteins: gp120, Gp41 5, Both proteins are on the outer envelope, essential for initiating replication cycle.

Answer 183

1. Gp120 on HIV surface, interacts with CD4 receptor on human cells, alongside co-receptors such as CCR5 or CXR4 2 Causes fusion between virus & cell surface, uncoating process occurs. Nucleocapsid is delivered into cytoplasm, releasing viral RNA & viral proteins. 3. Proteins convert single strand RNA into DNA, which interacts with integrase to for pre integration complex 4. Transported into nucleus by integrase. Causes negative effects in T cells. 5. Undergoes transcription & translation to produce more viral RNA as a polypeptide chain 6. Protease enzyme breaks the chain into HIV particles, which can infect cells

Answer 184

1. NRTIs & NNRTIs: Both target the HIV’s reverse transcriptase protein, preventing the conversion of viral RNA into viral DNA. 2. Integrase Inhibitors: Protease inhibitors 3 targets the Integrase, preventing the transfer of viral DNA into our DNA. 3. Protease inhibitors: Prevents the long polypeptide from being chopped, meaning no new infectious particles are released. 4. Fusion inhibitors: block action of Gp120 & Gp41, stopping receptor changing conformation 5. CCR5: prevents the co-receptor from engaging the virus, preventing entry into the cell, by blocking the co-receptor.

Answer 185

1. Lipinski’s rules do not apply to this drug. Must be given by IV infusion due to poor bioavailability. 2. Blocs entry of HIV into CD4 positive T cells. 3. Mimics Gp41, so it displaces the components of GP41, preventing normal fusion.

Answer 186

1) HIV approaches a host CD4+ T-cell. The viral membrane contains trimeric glycoprotein spikes. Each spike contains a gp41 and gp120 subunit. 2) Fusion begins with the binding of gp120 to the CD4 & chemokine receptors on the cell membrane. 3) Binding induces a conformational change in gp120 moving it aside & exposing gp41. 4) Fusion is mediated by gp41, which contains two heptad repeat domains, HR1 & HR2. 5) As gp41 is exposed, the hydrophobic terminus of gp41 embeds itself into the cell membrane. The HR2 domain (blue) begins to coil into the grooves on the HR1 domain of gp41 (red). This process, called zipping, destabilizes both cell & viral membranes causing a fusion pore in both membranes. This allows the HIV capsid to pass through the cell membrane and infection occurs.

Answer 187

1. Fuzeon is similar amino acids sequence to HR2 of 41, 2. Fuzeon is a peptide mimic of the HR2 region of gp41. 3. As a result, Fuzeon binds to the HR1 region. 4. Zipping cannot take place, and so infection is blocked.

Answer 188

CCR5 Inhibitors: 1. The drug, Maraviroc, blocks attachment of HIV to CCR5 receptor, halting HIV replication. 2. CCR5 inhibitors only effective in blocking the CCR5 receptor site. 3. However, HIV that uses CXCR4 receptors will be unaffected, meaning replication can continue. 4. Simply, CCR5 inhibitors will only be effective in people with HIV that uses the CCR5 receptor for replication.

Answer 189

1. HIV uses host cells’ genetic material to make viral DNA (reverse transcription). 2. Next virus integrates its genetic material into genetic material of host cells. 3. Done by enzyme called integrase 4. Integrase inhibitors block this enzyme & prevent the virus from adding its DNA into the DNA in your CD4 cells 5. Preventing this process prevents the virus from replicating & making new viruses. 6. Integrase needs amino acids to interact with metal ions, which bind to phosphate & break DNA so viral DNA can be inserted. 7. Inhibitors binds to the metal ions, preventing the breaking of our own DNA

Answer 190

Incorporated into growing DNA chain after phosphorylation, but has no hydroxyl group, meaning no new nucleotides can be added.

Answer 191

Non-nucleoside analogue reverse transcriptase inhibitors (NNRTI’s) inhibit viral DNA replication by binding at the allosteric non-bonding site of RT, causing a conformational change of the active site, this prevent the reverse transcriptase from moving, meaning it is unable to perform reverse transcription.

Answer 192

Protease inhibitors: 1. Protease inhibitors block the chopping up of the long polypeptide 2. Prevents small active viral proteins forming 3. Acidic residues in protease active site activates water which hydrolyses peptide backbone 4. Important to 1st attack hydroxyl group at carbonyl 5. Forms transition state bound tight to protease, tighter than peptide 6. Prevent enzyme binding to peptide and breaking the polypeptide

Answer 193

1. Resist to individual anti-HIV drugs develops rapidly during treatment (drug-induced resistance). 2. High levels of resistant strains are then observed in treatment naïve patients. 3. Treatment of HIV with a single anti-viral agent (monotherapy) offered only limited effectiveness.

Answer 194

1. Multidrug therapy, 3 different drugs from 2 different classes 2. Drugs attack virus at different points of replication 3. Sig reduce resist rates as hard to adapt to due to multiple areas of attack 4. Effective, less drugs needed, lowered side effects.

Answer 195

1. Increased liver disease from drug 2. Live past where HIV would kill them, but drugs damage liver overtime

Answer 196

1. When exposed to a pathogen for 1st time, we have no specific IgG antibodies against the pathogen. 2. As a result, infection can progress to disease. 3. Over time, we will produce antigen specific IgG antibodies, i.e. memory immune response. 4. The next time we are exposed to this pathogen, we will be protected from disease. 5. If exposed non-disease pathogen our bodies response would simply be the generation of immune memory.

Answer 197

1. Global Polio vaccination given by WHO 2. Polio almost entirely eradicated due to Herd immunity 3. R value, number of individuals infected by original infected person 4. No mass vaccination: Each host in contact with infected host becomes infected (with a certain probability) 5. With mass vaccination: Outbreak attenuated (or averted) by lack of susceptible hosts 6. Herd immunity threshold – proportion of population that needs to be vaccinated to prevent disease outbreak

Answer 198

1. Some diseases have multiple vaccines developed to target them. 2. A single disease may have vaccines from several different types, ie. inactivated, live-attenuated & recombinant. 3. The different types of vaccines can target bacterial & viral diseases.

Answer 199

1. Made from wild virus or bacteria. These are attenuated or weakened by repeat culturing. 2. To produce immune response, attenuated vaccines must replicate in person, so small dose is given 3. Immune response to live attenuated vaccine identical to natural infection 4. e.g. mumps, measles Pros: Cheap, adjuvants unnecessary (stimulates response alone) Cons: Potential cause of pathology, difficult to keep stable in storage/transport

Answer 200

1. Produced by growing bacteria or virus in culture media then inactivating with heat &/or chemicals. 2. Inactivated cannot replicate, whole dose of antigen is administered in the injection. 3. Generally, 1st dose doesn’t produce protective immunity, but “primes” the immune system. A protective immune response develops after the second or third dose. 4. E.g. Hepatitis A, Influenza Pros: Safer, more stable Cons: Costly, hypersensitivity

Answer 201

1. Sub-unit vaccines are comprised of one antigenic part of the pathogen. 2. Produced recombinantly, so other microbes produce desired antigenic component. 3. Antigenic components are often proteins, or surface polysaccharides. 4. A unique type of inactivated subunit vaccine is composed of long chains of sugar molecules that make up the surface capsule of bacteria. 5. Immune response to a pure polysaccharide vaccine is T-cell independent, means these vaccines stimulate B-cells without assistance of T-helper cells. 6. DNA vaccines are a type of recombinant vaccine. 7. e.g. Eg. Hepatitis B

Answer 202

1. Toxins: major virulence factors for some bacteria which cause disease. 2. Toxoids: chemically altered toxins, no longer pathogenic 3. Antibodies produced in response, resulting from toxoid administration neutralize the toxic moiety produced during infection rather than act upon the organism itself. In general toxoids are highly efficacious & safe immunizing agents 4. E.g. Tetanus, Diphtheria

Answer 203

1. Prepared from extracted cellular fractions e.g. meningococcal vaccine from polysaccharide antigen of the cell wall 2. Polysaccharides chemically attached to carrier protein to illicit T-cell response. Carrier proteins can often be toxoids, such as Tetanus toxoid. 3. High efficacy & safety 4. E.g. Pneumococcal, meningococcal

Answer 204

Monovalent (univalent) vaccine: immunize against 1 antigen or pathogen e.g. chicken pox

Answer 205

Multivalent (polyvalent) vaccine: immunize against 2 or more strains of one microorganism or multiple microorganisms e.g. DTP vaccine

Answer 206

1. Pathogen passaged several times under stress conditions. 2. Stress conditions pressure the virus to replicate slower & causes loss of virulence factors 3. Produces attenuated virus, no longer disease causing. 4. Virus is then purified & formulated.

Answer 207

1. Whole pathogen grown & killed e.g. Heat, chemical modification (formaldehyde) 2. Advantage: Relatively easy, Generally safe to administer - no risk of reversion, infection

Answer 208

1. Identify gene for the sub-unit being targeted. 2. Insert pathogenic gene into host-organism for production 3. Express protective antigen in safe easy-to-grow organism 4. Purify the antigenic sub-unit & formulate

Answer 209

1. Toxins isolated from pathogen & purified. 2. Toxins converted to toxoids (inactivated toxin) by chemical treatment. 3. Toxoid is then formulated.

Answer 210

1. Surface polysaccharide from pathogen is grown & isolated. 2. Carrier protein grown separately (usually in E. coli) & purified 3. Toxins are removed 4. Carrier protein & polysaccharide are covalently attached together (chemical crosslinking). 5. The ‘conjugate’ is then purified & formulated

Answer 211

1. Pathogen 2. Adjuvant stimulates better immune response 3. Antibiotics prevent bacteria growing in vaccine 4. Stabilisers longer shelf life 5. Preservative 6. Trace components

Answer 212

1. Enhances immune response, causing improved immune memory 2. Antigen-sparing: induces protective antibody response with less antigen 3. Cross-reactive immunity: induce “broader” immune respone 4. Overcome weakened immunity

Answer 213

Public health: All organized measures to prevent disease, promote health, & prolong life among the population as a whole

Answer 214

If vaccine efficacy is 80%, then of a vaccinated population 80% fewer people will contract the disease when they encounter the virus.

Answer 215

Herd immunity = indirect protection from an infectious disease due to high levels of immunity in the population

Answer 216

Herd immunity threshold: proportion of immune individuals in a population above which the incidence will decline.

Answer 217

Basic reproductive number (R0): average number of secondary cases arising from a typical case in a totally susceptible population

Answer 218

1. The β-lactam antibiotic is thought to mimic the D-Ala-D-Ala dipeptide involved in the cross-linking of peptidoglycan. 2. The β-lactam ring is highly strained (makes the β-lactam bond electrophilic). 3. The β-lactam antibiotic behaves as a substrate mimic for the penicillin-binding proteins (PBPs) & undergoes an acyl-substitution reaction with an active site nucleophile (usually a serine side-chain). 4. A stable acyl-enzyme complex is formed – blocks enzyme active site & removes nucleophile (irreversible inhibition). 5. The exact details depend on the specific antibiotic & bacteria involved.

Answer 219

1. Enzyme containing acyl-intermediate is inactive. 2. Results in bacterial lysis (requires actively growing cells). 3. High levels of antibiotic result in reduced lysis (‘The Eagle Phenomenon’). 4. Compare this reaction to that of serine proteases such as chymotrypsin.

Answer 220

1. Target modification (PBPs) – important for methicillin (a ‘β-Lactamase-stable’ penicillin). Results in methicillin-resistant Staphylococcus aureus (MRSA). 2. Amidases (removes penicillin side-chain). 3. β-Lactamases – perform acyl substitution reaction, making drug inactive

Answer 221

1. β-Lactamases mediate most of the resistance to penicillins & other β-lactam antibiotics. 2. Most β-lactamases use an active site serine (or cysteine) residue to perform the acyl-substitution reaction, which rapidly inactivates drug.

Answer 222

1. Genes for β-lactamases can be encoded for by bacterial chromosomes (cephalosporinases) or more commonly plasmids (small circular bits of DNA that can self-replicate) (penicillinases). 2. Plasmids can be transfected between members of the same bacterial species or sometimes different species – resistance can spread very rapidly.

Answer 223

1. Developing new antibiotics 2. Taking whole course of antibiotics 3. Careful use of antibiotics required 4. Change antibiotic’s structure: Temp. solution due to rapid development of resistance 5. Co-administer a blocking antibiotic: Level of protection can be poor & can result in resistance to multiple antibiotics, but sometimes successful e.g., co-fluampicil 6. Use a suicide inhibitor with the antibiotic e.g., clavulanic acid: Inhibitors undergo catalysis to generate a reactive species that inactivates the β-lactamase.

Answer 224

1. Semi-synthetic penicillin made by chemical acylation of 6-APA. 2. 6-APA produced in culture when no side-chain precursor is present. However, 6-APA is zwitterionic, ergo difficult to extract. 6-APA is often made from benzylpenicillin, as it’s solvent extractable. 4. Penicillin levels increased by changing culture media (side-chain precursors) & mutation of Penicillium fungi using UV radiation, mutagens etc. followed by screening for higher producing isolates. 5. Potential for contamination

Answer 225

1. Cephalosporins & cephamycin are alternative β-lactam antibiotics. 2. They bind to “Penicillin-Binding Proteins” (PBPs). 3. Used when infection is penicillin resistant 4. Made from penicillin N 5. Cephalosporins and cephamycin are made from penicillin N by Cephalosporium fungi and Streptomyces bacteria. 6. Semi-synthetic cephalosporins & cephamycin made by using acylase to remove the side-chain to give the cephem core.

Answer 226

1. Cephalothin undergoes rapid metabolism (removal of acetyl group). 2. Cephaloridine causes nephrotoxicity (kidney toxicity). 3. Cephalexin is usually preferred as it’s almost completely absorbed from the intestines despite having lower antibiotic activity.

Answer 227

1. Cephalosporinases catalyse hydrolysis of cephalosporins. 2. Endocyclic double bond migrates (when leaving group is present). 3. Similar reactions occur with cephamycins.

Answer 228

e.g. Sulbactam, Clavulanic acid 1. Each inhibitor behaves as a substrate, reacting with the penicillinase active site Ser residue. 2. Rearrangement of acyl-enzyme intermediate results in enzyme inactivation. 3. These inhibitors are examples of ‘suicide substrates’. 4. All of these drugs are good inhibitors of β-lactamases in vitro. 5. Most do not work well in vivo due to rapid inactivation and the difficulties in penetrating the bacterial cell wall.

Answer 229

1. Clavulanic acid is an irreversible inhibitor of class A β-lactamases (penicillinases). 2. It has weak antibiotic activity on its own, but it potentiates the effect of penicillin (prevents degradation by β-lactamases). 3. Normally co-administered with amoxycillin – coamoxyclav 4. Ineffective against class B (metal-dependent) β-lactamases (e.g., NDM-1) as they do ‘n’t use an active site residue behaving as a nucleophile. 5. Ineffective against class C (cephalosporinase) or D (extended spectrum) β-lactamases (does not bind to the enzyme). 6. Use of co-amoxyclav partly over-comes bacterial resistance to penicillin. 7. Use of clavulanic acid resulted in selection of bacteria producing Class A β-lactamases which were not inactivated by it, & had decreased catalytic efficiency of the β-lactamase. 8. Class B, C, D β-lactamases are not inactivated by clavulanic acid. These enzymes are less wide-spread than the Class A penicillinases, but their contribution to resistance could become much more significant.