Vaccines Fundamentals PDF

Vaccines Fundamentals of Vaccine Immunology Antigen Recognised by receptors. FoundonPathogen Epitope Area on antigen recognised by receptors. Immunogen Elicit immune r...

Vaccines Fundamentals of Vaccine Immunology Antigen Recognised by receptors. FoundonPathogen Epitope Area on antigen recognised by receptors. Immunogen Elicit immune response. Adjuvant Enhances antigen-specific immunity. Pattern Recognition Receptors (PRRs)  In membranes of cells that are part of innate immune system.  Not specific. IPAMPformanypathogens  Bind to pathogen associated molecular patterns (PAMPs)  PRR + PAMP = immune response PAMPs  Only produced by microbial pathogens  Survival/pathogenicity  1 PAMP for many pathogens, therefor, not specific Antigen Presenting Cells (APCs)  Part of innate immunity  Bridge innate and adaptive immunity  Antigen presented with MHC cellsurfaceofhost  Macrophages, B cells, dendritic cells presentsantigen to T cells T Cells  Only recognise antigens when bound to major histocompatibility complex (MHC)  MHC on cell surface of host cells  Recognisespecificantigens f Adaptive Immune Response humoral cell mediated  B Cells - Antibody mediated (humoral) immunity  T Cells – cell mediated immunity B Cells: T cell-independent response NI memory  B cell directly binds pathogen, proliferate & differentiate into plasma cells and produce IgM (1st antibody to be produced)  IgM – short lived & big (doesn’t bind well) not so great T cell mediated response somaticHypermutation Isotypeswitching  Goal of immunization  Somatic Hypermutation: B cells mutate and become better suited for antigen by increasing antibody affinity for antigen  Isotype Switching: allow antibodies to switch (IgM  IgG) to adapt to the need of the immune response IgG – most important for vaccines Immunological Memory  Memory cells  secondary immune response  T cell-independent immune response has NO memory Innate VS Adaptive Immunity Innate: Adaptive: Humoral Teenindependent simpatientation  1st line  2nd line cellmediatedTestifeediated stitling  Not specific (PAMPs)  Recognise specific antigens  Recognise PAMPs  Response improves in repeated  Response doesn’t improve exposure  No memory  Memory Tcellmediated Stimulation Of Immunity By Vaccines  Body detects threat  Initial detection stimulates innate immunity  Immune systems recognises epitopes on antigens  APCs – engulfment of antigens  Antigen with MHC protein inserted on surface of APC Concept of Vaccination  Harmless version of pathogen  memory cells but no pathological sequelae  Immune system  secondary immune response with strong & immediate protection  Humoral responses specifically enhanced  Memory Types of Vaccines Whole Vaccines Live Attenuated  Avirulent but antigenic  Lost capacity to induce full-blown dx  Effective and long lasting immunity  Doesn’t require multiple doses  Limitation: fragile, mutations, immunosuppressed C/I  Viruses: oral polio, measles, mumps, rubella  Bacteria: BCG Bacillus Calmette Guerin Killed/Inactivated  Killed by heat or formalin (won’t multiply)  Less effective & boosters needed  Viruses: inactivated polio vaccine Fractional Vaccines Subunit  Targets what’s pathogenic and eliminates side effects  Advantages: Stable, less side effects, cheap  Disadvantages: not as immunogenic, boosters  Examples: acellular pertussis and influenza Toxoid  Targets toxin produced by micro-organism that causes the dx  Adjuvant used to increase vaccine potency  Examples: diphtheria, tetanus Polysaccharide  Pure polysaccharide – T cell-independent (pooper) o B cells  IgM o No memory o Not consistently immunogenic in < 2 years  Conjugate polysaccharide – attached to protein o T cell activation & high affinity antibodies o Memory B cells o Examples: haemophilus influenzae, streptococcus pneumoniae Recombinant  Only epitopes  DNA sequence for antigenic protein inserted into expression system  Produces large quantities of specific antigen  Examples: hep B, HPV New Vaccines Nucleic Acid  Genetic material of pathogen stimulates immune response  DNA/RNA  host cells  antigens  immune response   Examples: covid-19 Vector Based  Modified virus delivers genetic code for antigen  Body’s own cells to produce antigen  Strong immune response Application Route of Administration  Deep subcutaneous or intramuscular route  Oral route (Sabine vaccine)  Intradermal route (BCG vaccine)  Scarification (small pox vaccine)  Intranasal route (live attenuated influenza vaccine) Adjuvants  Improves immunogenicity  Liberation of antigen, chemoattraction, inflammation  E.g. aluminium salts Role of Laboratory in Diagnosis of Infectious Dx Spheres of Activity  Clinical consultations   Direction of infection control  Prevention  Diagnostic microbiology laboratory Role of Clinical Laboratory  Diagnosis  Molecular models = pathogen detection & infection control  Antimicrobial susceptibility testing Essential Information  Patient details  Relevant clinical history  Specimen and source  Date of admission  Antimicrobial agents patient is on  Indication  Time and date Bacteriological Examinations  Value depends in correct collection of specimen and promptness with which it is sent to lab Type of Specimens Sterile: Non-sterile:  Blood  Pus  CSF  Sputum  Joint fluid/aspirate  Urine  Peritoneal fluid  Stool  Line tip Swabs  Moistened with 0.85% NaCl2 sterilesaline drygauze  p Eye, nose, throat, ear, skin, wounds, abscesses 701alcoholhypochlorite  Special transport media for: Neisseria gonorrhoea, chlamydia, ureaplasma, mycoplasma cultureoforganismsthatreq or togrow Espinrespiratorytractinfections streppneumonia Anaerobic Culture Pseudomonasaeruginosabacillusspecies Ecoli Suitable  Sinus aspirate  Capped syringe/closed sterile  Tympanocynthesis fluid tube  Lung biopsy  Tissue & pus  sterile container  Transtracheal aspirate Not Suitable  Urine  Wounds/sputum  Throat swabs Principles of Specimen Collection  Before antibiotic tx  Little contamination 7  Sterile equipment and aseptic technique  Clearly label container  ID source wherethespecimencamefrom  Sealed plastic bags sentto lab  Intact skin – 70% alcohol/hypochlorite disinfectant  Wound swab – sterile saline, using dry gauze  Leaking specimens are not only unsuitable for bacteriological culture but are potentially hazardous (red bio hazard stickers). Blood Culture  Gloves – universal precaution  Disinfect venepuncture site & stoppers of culture bottles with 70% OH  Disinfect skin with 70% isopropyl/ethyl alcohol Bone Marrow  Sterile container  Same as for blood culture Tissue  Sterile containers with small volume of sterile water or 0.9% saline  No formalin NB Urine Asymptomatic Patients 3 consecutive early morning specimens Bilharzia Ova Fresh urine between 12pm – 2pm Mycobacterial Culture 3 consecutive 1st morning specimens Culture Media  Provide nutritional components that bacterium gets in natural habitat  Water, carbon/energy, nitrogen, trace elements, & growth factors  Optimum pH, O2 tension, & osmolarity Why Bacteria Are Cultured  Diagnosis & drug susceptibility   Indication of role in dx process  Role as etiological agent  Antigens for vaccines Analytical Profile Index (API)  Wide range  Plastic strips  20 mini tests  Simple, rapid, reliable TB in Laboratory  Microscopy, culture, & sensitivity  M/E – fluorescent test & ZN stain o ZN stain – TB acid fast bacilli  Culture  Sensitivity – hain test (PCR for drug susceptibility) & GeneXpert Specimen – Sputum  2-3 early morning  Deep couch or induction  Other – lung biopsy, lung aspirate, NG aspirate Molecular Diagnosis  Suitable specimens – CSF, sterile body fluid or tissue  GeneXpert – sputum/endotracheal aspirate Yeast & Fungi  MCC&S Microscopy Culture Sensitivity  ID & susceptibility – candida spp  Pneumocystis jiroveci – Florescence test Specimens  Sputum – 3 early morning  Lung biopsy/aspirate  Biopsies and tissues  Dermatophytes – between 2 sterile glass slides Pneumocystis Spp  Induced sputum  Bronchial washing  Lung biopsy  3-4ml Malaria  2 thin, 2 thick smears  5ml EDTA blood Antimicrobial Susceptibility Testing (AST) Test bacteria against appropriate antibiotics to know which drugs are most active against the pathogens. Goal of Standardisation  Optimise growth conditions  Maintain integrity of antimicrobial agent  Reproducibility and consistency Methods of AST Quantitative: MIC, mg/l Qualitative: S, I ,R  Agar dilution  Disk diffusion  Broth dilution - Microtitre - Macrotitre Broth Dilution  Tubes increase in [antibiotic]  Incubated for 18hr at 37 degrees  Tedious Disk Diffusion  Antibiotic on filter paper disk  Standardised inoculum of test organism swabbed in MH agar plate  Results – Zone of growth inhibition or no zone Epsilometer (E-test)  Thin plastic strip with antibiotic concentration gradient  Elliptical inhibitory areas  MIC determined where growth ellipse intersect E-strip Antimicrobial Agents  Microbe: causes infection   Host Defence: fights the infection (sometimes not enough)  Antimicrobial Agent: helps eradication Origin Chemotherapeutics  Man-made  Para-aminobenzene-sulphonamide Antibiotics  Microbe-made  Penicillin Selective Toxicity Antimicrobial action affects processes in microorganism only and leaves host tissue in tact. Antibiotic Classification  Target site cellwallproteinnucleicacidsynthesis cytoplasmicmembranefunction  Bacteriostatic (inhibits bacterial growth) or bactericidal (kills bacteria)  Concentration dependent or time dependent Target Sites of Action Antibiotics may act on bacterial: 1. Cell wall synthesis BetaLactams Glycopeptides 2. Protein synthesis 3. Nucleic acid synthesis/modification of nucleic acid structure sulphonamides atrimethoprim 4. Cytoplasmic membrane function polymixins polyenes Cell wall synthesis Bacterial Cell Wall Structure:  Gram positive (GPB) or gram negative (GNB)  Peptidoglycan – hexose sugars and amino acids   GPB – thick layer  GNB – thin layer with outer membrane Cell Wall Synthesis:  Peptidoglycan is the target for selective toxicity  Peptidoglycan synthesis in cytoplasm Antibiotics: Beta-Lactams:BindPBP'scell wallcrosslinking Glycopeptides: BindDaiaDala ngeynsynbgunit.sn  Penicillin Pls  Vancomycin  Cephalosporins come  Teicoplanin  Monobactams Make  Carbapenems cakes 1. Beta-Lactams  Distinguished by ring attached to B-lactam ring and side chains  MOA: binds to penicillin-binding proteins (PBPs) which are responsible for final stages of cell wall cross-linking Beta Lactam-Beta Lactamase Inhibitor Combinations:  Beta lactamase – enzyme inactivating B-lactams  Beta lactamase inhibitors – inhibit B-lactamase & therefore given with antibiotics Amoxycillin Clavulanate Augmentin + = Piperacillin Tazobactam Tazocin + = 2. Glycoproteins  Bind to D-ala-D-ala  Prevents new subunits  growing wall   Act at earlier stage than B-lactams  Examples – vancomycin & teicoplanin Opeptidyl transferase peptidebonds Protein synthesis aminoacylRNA  Antibiotic prevents aminoacyl RNA from entering acceptor sites on 30s  Blocks action of peptidyl-transferase and thereby prevents peptide bond synthesis  Binds 23s and 5s & blocks translocation step A toughchallenge can make frustrations Antibiotics:  Aminoglycosides (gentamicin) bactericidal serious gramneg infections  Tetracyclines (doxycycline) broadspectrum atypicalbacteria Lymedx  Chloramphenicol broadspectrum serious infections meningitis  Clindamycin softtissueinfection MRSA anaerobes pneumonia  Macrolides (erythromycin) respiratorytract infections  Fusidic acid narrowspectrum skininfectionscausedbystaphylococcus Nucleic acid synthesis/modification of nucleic acid structure Sulphonamides Compete with PABA for dihydropteroate synthetase enzyme. acidsynthesis inhibitsfolic Trimethoprim Compete with dihydrofolic acid for DH reductase enzyme.  Work together  Sulphonamide blocks para-aminobenzoic acid (PABA)  dihydrofolic acid  Trimethoprim blocks dihydrofolic acid  tetrahydrofolate PABA  DH acid  tetrahydrofolate  purines + pyrimidines sulphonamides Trimethoprim Quinolones: Modification of nucleicacidstructure  Inhibits DNA gyrase o DNA gyrase: regulates super helical structure of bacterial DNA   Promotes cleavage of bacterial DNA  Rapid death  From nalidixic acid (older drug) Cytoplasmic membrane function  Anti-GNB  Free aa groups act as cationic Polymyxins detergent that disrupts phospholipid structure  E.g. colistin Polyenes  Anti-fungal Other Antibiotics  Metronidazole  Goes after bacterial DNA Lower UTI Agents Nalidixic Acid uncomplicatedUTI  Quinolone therefore inhibits DNA gyrase  GNB  Resistant: pseudomonas  use another quinolone  Indication: uncomplicated UTI  C/I: lactation Nitrofurantoin uncomplicated UTI prophylaxis  Inhibits enzyme systems in bacteria  Wide spectrum – GNB + GPB  Resistant: pseudomonas & proteus spp piperacillin  Indication: prophylaxis, pregnancy, & uncomplicated UTI Fosfomycin Uncomplicated lower UTI  Interferes in early step in peptidoglycan formation & thus cell wall synthesis  GNB + GPB  Uncomplicated lower UTI Spectrum of Activity of Antimicrobials Penicillin G/Benzylpenicilin G  URTI, syphilis, meningitis  Streptococci (meningitis), Neisseria meningitides, treponema pallidum (syphilis), and anaerobes Ampicillin  Same as penicillin G but more (broader spectrum)  Enterococcus species  Haemophilus influenzae  Enterobacteriaceae (s. typhi) Piperacillin  Same as ampicillin but includes,  Pseudomonas aeruginosa NB Cloxacillin  Active against – staph aureus  Not active against – methicillin resistant staph aureus (MRSAs) Cephalosporins  B-lactam  GPB cover decreases as you go up generations  GNB cover increases as you go up generations  E.g. 1st gen has good GPB cover but poor GNB cover 1st Generation (cephradine & cephalothin):  UTI, & Skin & soft tissue infections (SSTI)  Streptococci (not enterococci)  Staph aureus (not MRSA)  Enterobacteriaceae (e. coli, klebsiella pneumoniae, proteus mirabilis) 2nd Generation (cefuroxine):  Broader than 1st gen  UTI Cephamycin (cefoitin):  Different because it covers anaerobes  Surgical prophylaxis 3rd Generation (cefotaxime, ceftriaxone, ceftazidime):  Crosses BBB  Penetrates CSF therefore good for bacterial meningitis  STIs  Similar to 2nd generation 4th Generation (cefipime):  Pseudomonas aeruginosa – not covered by 3rd gen  Not active against anaerobes and enterococci Carbapenems  Very broad  Not active against: MRSA, stenotrophomonas maltophila Beta-Lactam + Beta-Lactamase Inhibitor Combos Amoxycillin + Clavulanic Acid = Augmentin  Community acquired organisms  GNB & GPB  Not active against: pseudomonas aeruginosa & ESBL producers Piperacillin + Tazobactam = Tazocin  Hospital acquired organisms  pseudomonas aeruginosa & ESBL producers Aminoglycosides (gentamicin & amikacin)  GNB (excl. anaerobes)  Used in combination (Synergy) Tetracycline  Against protein synthesis  Atypical organisms – rickettsia, chlamydia, mycoplasmas Chloramphenicol  Broad  Anaerobes, rickettsia, chlamydia, mycoplasmas  Not used so much in S. typhi anymore because of resistance Macrolides (erythromycin, clarithromycin)  Broad  Bordetella pertussis, rickettsia, chlamydia, mycoplasmas, legionella spp, treponema pallidum  Alternative to B-lactam allergy Clindamycin  Active against – streptococci (not enterococci), staph aureus (not MRSA), anaerobes  Inactive against – GNB Glycopeptides (vancomycin, teicoplanin)  Cell wall sunthesis  GPB  Only one that covers MRSA Linezolid  Same as glycopeptides  Not nephrotoxic Co-Trimoxazole  Atypicals – chlamydia, mycoplasmas  Increasing resistance Rational Use Of Antibiotics And Resistance Development Goal of Antibiotics  Clinical and microbiological cure o Clinical – patient gets better o Microbiological – microbe eradicated  Avoid side effects  Avoid resistance o Therefore choose agent rationally Best Antibacterial Agent  Proven efficacy  Narrow spectrum  Restricted distribution in body  Limited side effects  Simple dosage (patient must adhere)  Cheap Resistance Clinical Resistance No response and patient doesn’t get better. Min inhibitory concentration (MIC) of organism is Laboratory Resistance above breakpoint. What determines clinical resistance/response  Accuracy of diagnosis  Clinical status  Accuracy of laboratory diagnosis o Aetiological agent o Susceptibility test result Producinginactivating enzymes aactiveremoval Mechanisms of Resistance: cell modification envelope TargetModification MetabolicPathways Bypassing Producing Inactivating Enzymes Inactivating the drug before it even enters or shorty after entering the microbial cell. Beta-lactamase BSBL a carbapenases NOTinhibitedby B lactamaseinhibitors  Plasmids & chromosomes triggeredtoproduce  p Inducible or non-inducible Classic  Inactivate penicillin  Inhibited by B-lactamase inhibitors  Plasmids Extended Spectrum B-Lactamase  Inducible (ESBL)  Inactivated by B-lactamase inhibitors Broad Spectrum B-Lactamase  Chromosome (BSBL)  Slowly inducible   Doesn’t inactivate carbenems  Not inhibited by B-lactamase inhibitors  Chromosome, plasmids, & transposons Carbapenamases  Inhibit penems and carbapenems  Not inhibited by B-lactamase inhibitors Aminoglycoside modifying enzymes  Gentamicin group of inactivating enzymes  Amikacin group of inactivating enzymes  Miscellaneous enzymes Macrolide-lincosamide-streptogramin modifying enzymes  Inducible  Problem in S. aureus, S, pneumoniae, and S, pyogenes Chloramphenicol amino transferase  Problem in H. influenzae and anaerobes Cell Envelope Modification & Active Removal Envelope becomes less penetrable to drug. Drug is actively expelled.  Single – low level resistance  Combination – high level resistance  Cell envelope modification – single drugs or groups of related drugs  Active drug removal – single or related groups OR unrelated groups Target Modification Target site binds with less strength.   All drugs  B-lactams & quinolones  Multiple step mutations Mutant selection window  Longer the mutant in selection window  More time to multiply  Increase resistance  Increased change of next mutation Drugs needing infrequent administration Long half life  High peak & gradual declining slope  = resistance Slow release  Low peak & steady state  Prevents resistance Time vs Concentration dependent drugs Time Dependent Drugs Effect depends on time that [drug] is above MIC.  Cell wal synthesis inhibitors  Some protein synthesis inhibitors (macrolides) Concentration Dependent Effect depends on [drug] exceeding MIC. Drugs  Most protein synthesis inhibitors Bypassing Metabolic Pathways End product acquired in a different way. Community VS Hospital Acquired Infections Community  Before or within 48hrs of hospitalisation  No regular health care system contact Hospital  After 48hrs of hospitalisation  Regular contact with health care system Hospital Acquired Infections (Nosocomial Infections)  Faecal peritonitis after bowel surgery  Blood stream infection due to colonised central venous line  Catheter associated UTI iatrogenic  Latter 2 are iatrogenic infections  Pseudomonas aeruginosa Mycobacterium Tuberculosis & Non-Tuberculous Mycobacterium Need to know: sear SKA What is TB? formingnonmotilebacillusw Aerobicnonspore curved beadedrod shapedmorphology How is it transmitted? Airborne dropletnuclei How is it diagnosed in the lab? Treatment? Introduction Aerobic, non-spore-forming, non-motile, bacillus, with curved and beaded rod- shaped morphology.  Pulmonary/extra-pulmonary TB  Airborne – droplet nuclei  Slow development, chronic, & granulomatous response  Hardly cultures – takes 8 weeks  Hardy surviveinmanyconditions Cell Wall  Gram positive  Mycolic acid + lipoarabinomannan (LAM) o Lipopolysaccharide (LPS) in GNB  Hydrophobic  slow growth  drug resistance Implications For Microscopy  Heat/detergents aid dye to penetrate cell wall  ZN – basic fushin dye  Fluorochrome – fluorescent dye Disease Transmission ptuberclebacilli  Person-to-person via airborne transmission (droplet nuclei) in confined environment  10 bacilli  infection  Indoors, dark, damp spaces  Direct sunlight kills MTB   Larger droplets ineffective as they just fall to the floor  Small droplets reach alveolar spaces  HIV patients aren’t more infective – smear negative Infectiousness Most Infectious Smear positive & lung cavities Less Infectious Smear negative HIV Least Infectious Extrapulmonary & latent TB Latent TB Infection 94yg.EEy'mlephages  Breathes in TB bacilli but immune systems keeps it under control epithelioidcells cost  Not infectious & asymptomatic  Mantoux tuberculin skin test (TST) or interferon-gamma release assays (IGRAs) but doesn’t differentiate between latent TB & RB Dx Institutional Spread  Hospitals Overcrowding is the  Homeless shelters biggest contributor.  Correctional services Sites of TB Dx  Pulmonary – lungs  Extra-pulmonary  Miliary – bloodstream (carried to all parts of body) Pathogenesis  Droplet nuclei (containing tubercle bacilli) float in the air for several hours 1. Droplet nuclei inhales  alveoli 2. Multiply in alveoli 3. Bacilli enter bloodstream and spread throughout body  4. 2-8 weeks macrophages surround bacilli a. Keep it latent MTBUrease acidification b. Bacilli multiply rapidly = TB Dx Bacillus survives Antioxidants detoxify Immunity  MTB survives in macrophages  MTB urease – prevents acidification of phagosome  Antioxidants - detoxify reactive oxygen species from phagosome Cell Mediated Immunity (against MTB)  Cell mediated immunity in 3-9 weeks kills bacteria  µ Activated macrophages accumulate lytic enzymes  mycobactericidal  Epithelioid cells (highly stimulated macrophages) & CD8+ lymphocytes Caseous Necrosis & Cavitation  liquify & discharge via bronchial tree Caseous Necrosis  102 – 104 organisms Tissuedeaththatlookslikecheese  increased mycobacterial replication Cavitation  107 – 109 organisms cavitiesforminlungs advanceddx Clinical Diagnosis  Chest Xray  strong presumptive diagnosis  Positive sputum smear Signs & Symptoms  Asymptomatic (latent) infection progresses to constitutional symptoms oyghingup a C o Anorexia, fatigue, weight loss, chills, fever, & night sweats Haemoptysis – advanced dx  Sudden massive haemoptysis – pulmonary artery eroded by cavitation – fatal  Productive cough > 2 weeks Tuberculin Skin Test (TST)  Mantoux test  Children  Hypersensitivity to tuberculoprotein  Positive – MTB & BCG vaccination ÉiiÉÉreclauiaisolia Laboratory Diagnosis ééié iii Microscopy canfluorescent IDacidfastbacilliAFB confirmatory tests  1st line of diagnosis in SA – Xpert Ultra (NAAT) NBinareas w highprevalence ofMDR TB  TB culture – gold standard Simultaneouslydetectsrifampicinresistance  Microscopy – susceptible TB  Smears  Multiple deep couch specimens  Timeous transport  Delays  decreased viability of mycobacteria & overgrowth by flora  Cold chain (4 degrees) Specimen Collection  Cover mouth while coughing  Outside  Away from people  Don’t stand near patient or stand behind them Microscopic Appearance: ZN Stain  Mycobacteria are acid fast bacilli (AFB)  Pink/red organism on blue background  Irregular beading appearance MTB Culture  Gold standard  More sensitive  10 AFB per ml  Slow growing on solid media (6-8 weeks)  o Buff-coloured (never pigmented) o Bread crumbs or cauliflower appearance  Faster in liquid growth media (42 days) 6 week o Show cording Mantouxunreliable Harderto diagnose or nonspecificsmearnegativeotherinfectionspresent HIV & TB bacilliinsputum Lessinfectious weakenedcough Diagnosis more difficult in HIV+ because:  Smear negative sputum  CXR abnormalities non specific  Other pulmonary infections present  Mantoux test unreliable Advanced HIV less like to transmit TB because:  Decreased cavitary dx  Decreased sputum bacillary load (bacilli not in lungs)  Weakened cough TB-IRIS (Immune Reconstitution Inflammatory Syndrome)  Immune function improves due to ARTs Tea stringly atheantigen'snsak.SE rE9iouTy9gis ve inflammation symptomsthat 1 saudede appear  Within 3 months of starting ARTs 9b mamytitsyeningsp.IE  Presents with unusual severity or unusually rapid progression  Confused with treatment failure Patient already on TB treatment clinically deteriorates Paradoxical TB-IRIS when started on ARVs. Patient already on ARVs present with undiagnosed Unmasking TB-IRIS TB. Previously undiagnosedTBbecomesapparentafterstartingARVs Non-Tuberculous Mycobacteria  AKA “environmental mycobacteria”  Soil, water, animals  Infection associated with defects in host defences  Multiple positive cultures to be clinically significant Transmission Cutaneous, respiratory, and GI routes AFT with no cording in culture. Lab Diagnosis Smear 12-18 months of multiple anti-mycobacterial drugs. Treatment Surgery (localised cutaneous infection). Mycobacterium Kanasii  Disseminated dx  Test for rifampicin susceptibility (R  clarithromycin/azithromycin)  Rifampicin treatment for 18 months Mycobacterium Marinum  Chronic cutaneous lesions  Open skin exposed to colonised water  Fish tanks/unchlorinated pools  Needs complete excision Mycobacterium Ulcerans  Buruli ulcer  Closely related to M. marium  Painless nodule progresses to deep ulcer  Surgery Mycobacterium Avium-IC Complex  Pulmonary dx – normal immunity  Disseminated dx – poor immunity (AIDS)  Cervical lymphadenitis – children  Rare sites like skin – hot tub ulcer Anti-TB Drugs Drug Susceptible Tuberculosis (DS-TB) 1st Line Anti-TB Drugs Abbreviation Drug MOA MOR INH Isoniazid Inhibits cell wall synthesis i RIF Rifampicin Inhibits RNA synthesis 1rpoB RNA Disrupts plasma membrane & 1pricA PZA Pyrazinamide energy metabolism Plasma EMB Ethambutol Inhibits cell wall synthesis 1 emb intensive 4for2 Standard Treatment Regimen Continuous 2for 4 Patient put on RIPE if:  never been on TB treatment  TB treatment < 4 weeks  Presumed DS-TB: 6 month treatment o Intensive phase – 4 drugs for 2 months o Continuous phase – 2 drugs for 4 months (INH, RIF) Rationale Behind Multidrug Treatment:  Infection with bacilli at different growth phases  Act simultaneously on all subpopulations  Some drugs act best in acid environment and other in alkaline  Prevents emergence  Duration of treatment reduced  Metabolically active – still growing within cavities  Intracellular – within macrophages  Semi dormant – slow replicators  Dormant Isoniazid (INH) disruptscellwallsynthesis  Highly bactericidal  T1/2 – 1-3 hrs  Additional pyridoxine – prevents peripheral neuropathy MOA  Activated by catalase-peroxidase countered by KatG  Inhibits enoylreductase inhA counteredby inna o Fattu acid biosynthesis (cell wall synthesis) MOR  Mutation in katG gene o Lower levels of activated INH o High level resistance  Mutation in inhA gene o Codes for enoylreductase inhA o Low level resistance Side Effects  Hepatitis – initiate after normal LFT  Increase level of epileptic drugs (phenytoin & carbamazepine) Rifampicin interferes w RNAsynthesis bybinding to RNA polymerase O genetranscription  Potent sterilizer – intra and extracellular bacilli  Lipid soluble  T1/2 – 2-3 hrs  Recycled by liver & excreted by faeces MOA  Bind to RNA-polymerase  Interferes with gene transcription MOR  Mutation of rpoB gene  Decreased affinity for RIF  +/- 90% RIF resistance also have INH resistance = MDR-TB Side Effects  GIT  Cutaneous reactions  Hepatitis  Colours urine, sweat, & tears pink (indicator of compliance) Drug Interactions  Break down other drugs more rapidly  Increase dose of contraception  Lowered by ART Pyrazinamide Disruptsplasmamembrane energymetabolism  Potent steriliser in IC acidic  Highly effective in 1st 2 months because it works well in areas of acute inflammation  Rapidly distributed  T1/3 – 10 hrs  Metabolised in liver & excreted in urine MOA  Activated by pyrazinamidaze  pyrazinoic acid  inhibit fatty acid synthesis  Disrupt plasma membrane & energy production  Bind to & affect ribosomes MOR  Mutation in pncA gene  Codes for pyrazinamidaze  lower levels of activated PZA Side Effects  Liver damage (hepatic impairment C/I)  Arthralgia (aspirin)  Rash on sun-exposed areas Ethambutol  Prevent/delay emergence of resistance  Excreted in urine & faeces MOA  Inhibits arabinosyltransferase that’s involved in cell wall biosynthesis  Static MOR  Mutations in emb gene  Codes for arabinosyltransferase Side Effects  Progressive loss of vision (retrobulbar neuritis)  Peripheral neuropathy C/I  Optic neuritis  Poor renal function  < 8yrs old can't tell you if their vision is worsening Drug Resistant Tuberculosis (DR-TB) Diagnosis made in lab not clinically. MDR-TB/RR-TB Rifampicin & isoniazid RR-TB Rifampicin Pre-XDR-TB Rifampicin, isoniazid, & any fluoroquinolone Rifampicin, isoniazid, any fluoroquinolone, & 1 XDR-TB group A drug Risk Factors  Exposure to DR-TB  Active TB in high prevalence drug resistance area  Positive sputum smear after 2 months of combo treatment  Travel to are of high drug resistance Primary vs Secondary Drug Resistance Primary Transmission of DR-TB from person to person. Secondary On treatment & resistance develops. Noexposure BPal C regimen MDR-TB Treatment Exposure 1month BPal C regimenbuttestedforresistance  6 months BPaL-L regimen (bedaquiline, pretomanid, linezolid, & levoflozacin)  Non-pregant & >15 years  MDR/RR-TB & pre-XDR-TB who haven’t had exposure to bedaquiline, pretomanid, and linezolid  Previous exposure >1 month will also be started on BPaL-L regimen but will be tested for resistance Bedaquiline  Dairyquinolone class  Inhibits ATP generation  Backbone of MDR/RR-TB Fluoroquinolones: Levofloxacin  Prevents DNA unwinding  inhibits nucleid acid synthesis  Kills actively multiplying organisms MOR:  Mutations in gyrA & gyrB genes  DNA gyrase decreases affinity for FQ  LABORATORY DIAGNOSIS OF VIRAL INFECTIONS Role Of Virology Laboratory  J Diagnosis – essential for dx where antiviral therapy is available o E.g. herpes, influenza, hepatitis, HIV  Monitoring – tracks response to therapy and identifies DR (molecular techniques)  Epidemiology – surveillance of viral dx outbreak including detecting atypical/emerging viruses Importance Of Diagnosis Clinical Management  Timely diagnosis influences management and prognosis  Rubella – during 1st trimester poses an 85% risk of congenial rubella syndrome CRS  Genital Herpes – may require C-Section to prevent neonatal transmission  HIV Diagnosis – therapeutic & prognostic decisions Public Health  Diagnosis prevents spread through blood banks o E.g. HIV, HBV, HCV  Facilitates vaccine development (e.g. influenza)  Containment strategies (e.g. haemorrhagic fever) Factors Influencing Viral Detection  Proper specimen collection timing  Collection from appropriate site  Effective & timely processing of specimen Viral Diagnostic Techniques Electron Microscopy & Viral Culture  Previously used for virus discovery & research   Not routine due to cost and time Serology  Detects antigens/antibodies  IgM – recent exposure  IgG – past exposure Indirect ELISA  Detects antibodies using serology plate with antibodies 999T Effie antigen binding  Followed by colorimetric detection Direct ELISA  “sandwich” technique Detectsantigensinasample  Detects antigens using capture and detector Lowsensitivity antibody  Enzyme-labelled colour change Competitive ELISA  Measures antibody levels based on competition Detectaquantifyantigen with enzyme-labelled antibodies HIV Diagnostic Methods HIV ELISA Advantages: Limitations:  Highly sensitive  Not for < 18 months willdetectmaternalantibodies  Tests large volumes  Specialised equipment  Shortens diagnostic window  Trained staff  Delayed results HIV Rapid Test Advantages: Limitations:  Quick (20 min)  Weak band interpretation may  Visible to patients cause false positives  No specialised equipment  Immediate counselling  Primary HC settings  Concerns about patient’s response Western Blot  Confirmatory test  Detects antibodies against specific viral proteins  Used when other methods had inconclusive results Molecular Virology Techniques Polymerase Chain Reaction Advantages: Disadvantages:  Rapid  Expensive  Highly specific and sensitive  Prone to contamination  Early diagnosis  Requires skilled staff  Monitors treatment Specimen Collection  Aseptic technique & universal precautions gloves  Timing – collected early in dx for optimal viral concentration  Site – taken from site containing the virus  Transport – quickly & in ice to maintain viral viability pagintigobialagentto contamination TransportedinVTM Sample Requirements By Clinical Syndrome Gastroenteritis stool Rotavirus & adenovirus Respiratory Infection Throat/nasal swabs Rhinovirus & influenza Throat/nasal swabs or Pneumonia/Croup RSV & influenza aspirates Encephalitis/Meningitis CSF Enterovirus & HSV Eye Infection Eye swab HSV & CMV Congenital Infections Blood, urine, or CSF Rubella & CMV Specimen Transport And Storage  Viral Transport Media (VTM) – ensures viability by preventing desiccation & maintaining proper pH  VTM Composition: o Essential nutrients (e.g. vitamins, glucose, amino acids) o Microbial agents to prevent contamination Key Points For Lab Diagnosis  Proper sample collection, timing, and transport are essential for accurate diagnosis. Ma chain  PCR and serological methods (ELISA) are the mainstays of routine viral diagnosis.  Molecular techniques like PCR are highly sensitive and allow for early detection but require expert handling to avoid false positives. it'sproneto contamination LABORATORY DIAGNOSIS OF HIV HIV In SA  17.1% adult prevalence  95-95-95 target o 90% know their status o 91% on ART  o 94% virally supressed Diagnostic Methods Initial Diagnosis HIV rapid test Adults Serology In-lab ELISA Serological can’t be used due to maternal Children < 18 months PCR antibodies Other Tests HIV Drug Resistance Test Patients with virological failure Plasma viral RNA monitoring HIV VL (especially on ART) Diagnostic Window  Period between HIV transmission and detection of measurable markers  Antibodies, antigens, or nucleic acids  Length determined by type of serological assay used and individual’s immune response  HIV RNA detectable after 2 weeks but false negatives may still occur HIV Testing Algorithms  Adults & children > 18 months – HIV rapid test as screening  Children < 18 months – HIV PCR 2-Test Approach (High Prevalence Areas)  1ST test (screening)  if reactive (positive)  another test for confirmation  1ST test (screening)  non-reactive (negative)  another test if recent exposure is suspected  HIV ELISA – indicated in case of discordant or indeterminate results HIV Rapid Tests  Lateral-flow (immunochromatographic) assays  Whole blood, serum, or saliva Advantages  Quick, easy, no lab required  Facility and community settings  Results in under 30 minutes Self-tests are encouraged to increase testing uptake (especially in hard-to-reach populations) and should be confirmed by trained healthcare professionals. Evolution Of Rapid Tests 1ST Generation  IgG  56 days 2ND Generation  HIV- 1/2 IgG (more specific)  42 days 3RD Generation  HIV-1 group M & O  HIV-2  IgG & IgM  22 days 4TH Generation  HIV-1 group M   HIV-1 P24  HIV-2 IgM & IgG  15-17 days 5TH Generation  HIV-1 antibodies  HIV-2 antibodies  HIV-1 p24 antigen In-Lab HIV Testing PCR  Molecular tests or HIV Western blot may be considered when rapid tests give discrepant results  ELISA – main serological HIV test  Very high sensitivity (low rate of false positives)  Very high specificity (low rate of false negatives) Early Infant Diagnosis (EID)  HIV nucleic acid PCR used 6w̅post stopping breastfeeding o Detects HIV-1 DNA/RNA  Maternal antibodies interfere with serological testing  Testing intervals: o Birth, 10 weeks, 6 moths, 18 months o 6 weeks post-cessation of breastfeeding HIV VL Testing  Quantitative real-time PCR  Measured amount of cell-free viral RNA in plasma  Monitoring ART efficacy  Diagnosing treatment failure Virological Failure VL > 1000 copies/ml after 2 years of treatment HIV DR Testing: Genotypic Resistance Testing  Detects mutations in HIV genome associated with DR  Based on PCR technology molecular   Cheaper and quicker than phenotypic testing  Results interpreted using algorithms (Stanford HIVDB) which assigns susceptibility scores Quality Assurance  SOPs – standard operating procedures (SOPs) can  Reduce testing errors and ensure reliability we go 551 aaoM VALIDITY OF LABORATORY RESULTS Evaluating A Laboratory Test  Validity – how well a test gives an accurate result, answering whether the test truly measures what it claims to  Comparison to a gold standard – new test’s accuracy is evaluated against a reference or gold standard test to determine its validity Key Metrics In Test Evaluation Sensitivity  Ability of a test to correctly identify true positives  Importance – test with high sensitivity minimises false negatives, ensuring dx are correctly identified 𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠 (𝑇𝑃) 𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦 = ideally 𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠 (𝑇𝑃) + 𝐹𝑎𝑙𝑠𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠 (𝐹𝑁) Specificity  Ability of a test to correctly identify true negatives  Importance – test with high specificity reduces false positives, ensuring those without the dx aren’t misdiagnosed 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠(𝑇𝑁) 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐𝑖𝑡𝑦 = 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠 (𝑇𝑁) + 𝐹𝑎𝑙𝑠𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠 (𝐹𝑃) Predictive Values Positive Predictive Value  Probability that the patient truly has the dx if the test is positive.  Influenced by sensitivity and prevalence of dx in population. 𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠 (𝑇𝑃) 𝑃𝑃𝑉 = 𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠 (𝑇𝑃) + 𝐹𝑎𝑙𝑠𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠 (𝐹𝑃) Negative Predictive Value  Probability that the patient truly doesn’t have the dx if the test if negative.  Influenced by specificity and prevalence 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠(𝑇𝑁) 𝑁𝑃𝑉 = 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠 (𝑇𝑁) + 𝐹𝑎𝑙𝑠𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠 (𝐹𝑁) Test Quality: A Critical Cycle Ordering A Test  Select appropriate lab tests based on clinical indications Methodology  Validated & standardises testing methods  Ensures accuracy and reliability Quality Assurance And Quality Control  Continuous monitoring to ensure consistent test performance Result Accuracy  Test should yield results – reproducible and reliable for clinical decision- making Clinical Decision-Making  Accurate tests  treatment plans  patient outcomes Practical Application  New diagnostic methods compared to gold standards – ensures validity  Test sensitivity & specificity evaluated  PPV & NPV considered based on population prevalence Summary  Laboratory test validity – clinical management, influencing diagnosis, treatment, and patient outcons  Key metrics (sensitivity, specificity, PPV, NPV) – helps assess reliability of results  Continuous quality assurance and adherence to standardised methods are vital to maintain test accuracy and reliability HIV VIROLOGY Basic Virology Of HIV: Structure, Genome, Characteristics HIV Classification  Family: retroviridae  Genus: lentivirus o HIV-1, HIV-2, Simian Immunodeficiency Virus HIV Structure  Spherical Virion  Enveloped  80-100 mm diameter  Diploid Genome  2 copies of single-stranded positive-sense RNA  9-10 kb  Capsid protein Core  p24 Gag  gp120 – surface protein – interacts with CD4 Envelope Proteins Env  gp41 – transmembrane protein – facilitates membrane fusion HIV Genome ViralmRNA viralproteins Encodes for: penzymes gpro gp pay  Structural proteins – Gag, Pol, Env precursorcleaved  Regulatory genes – Tat, Rev byprotease  Accessory proteins – Vif, Vpr, Vpu, Nef Structural Proteins Gag  Capsid p24  Enzymes Pol  Reverse transcriptase, integrase, & protease Env  Envelope proteins   Gp120  1p41 HIV Replication Cycle 1. Attachment & Entry  HIV attaches to CD4 receptors via gp120  Interaction with co-receptors (CCR5 & CXCR4)  gp41 mediate membrane fusion  viral entry CCR5 delta32 mutation resistance toHIV 2. Reverse Transcription  Viral RNA revere transcribe  double-stranded DNA  Reverse transcriptase highproneto error mutations 3. Integration  Viral DNA integrated into host genome by integrase  Forms a provirus  Long term persistence within host cell 4. Transcription & Translation  Host RNA polymerase transcribes proviral DNA  Viral mRNA translated  viral proteins 5. Assembly, Maturation, & Release  Viral proteins & RNA assemble at host cell membrane  Protease cleaves Gag & Pol precursor proteins during maturation  New virion buds from host cell completes replication cycle Host Immune Responses To HIV Initial Response Acute Infection  HIV transmitted to CD4+ T Cells & dendritic cells APC  Massive viral load increase (primary viremia) within 10 days Cytokine Storm & Seroconversion Syndrome  Antibodies detectable by day 10 using 4th gen ELISA  Detects p24 antigen & HIV antibodies Cell-Mediated Immune Response  CD8+ T cells  cytotoxic response to reduce viral load  CD4+ T cell levels initially recover  HIV persists in reservoirs & continually evades immune control Chronic Infection  CD4+ T cells become depleted  Viral load stabilises at set point  Immune exhaustion & susceptibility to opportunistic infections Host Factors Influencing HIV Pathogenesis Genetic Factors CCR5-delta32 Mutation  Homozygous carriers of 32-bp deletion  Resistant to HIV infections  Mutation prevents HIV from using CCR5 as co-receptor for entry HIV Genetic Diversity  High mutation rate due to error-prone reverse transcriptase  Heterogeneous population of viral genomes (quasispecies)  Immune escape and rapid evolution HIV Cure Strategies CCR5 Mutation-Based Cure  Stem cell transplants from donors with CCR5-delta32 mutation  Remission/cure by replacing patient’s immune cells with HIV-resistant cells Gene Editing  CRISPR/Cas 9  Introduce CCR5 mutations   Or target viral genes within infected cells  Block HIV replication Latency-Reversing Agents  Reactivate latent HIV in reservoir cells  Allows immune system to target and eliminate these cells Shock And Kill  Shock – reactivate latent virus  Kill – viral clearing using immune-based therapies Barriers To HIV Cure Latent Reservoirs  Reservoirs in resting CD4+ T cells  Lymphoid tissue  Difficult to eliminate since latent HIV isn’t recognised by immune system or ARVs variation Immune Evasion MHCI downregulation Glycosylation  HIV evades immune system through: o Antigen variation o Glycosylation – masking epitopes o Downregulation of MHC class I – escape cytotoxic T cells Persistent Low-Level Replication  Some viral replication persists even on ART  Contributes to ongoing immune activation & difficulty achieving complete cure Conclusion  Substantial progress has been made in understanding HIV virology & ongoing clinical trials are exploring potential cures.  Combination strategies targeting different aspects of the virus and its reservoirs is likely necessary for successful HIV eradication.  ART & RESISTANCE How To Prescribe ART ART Regimen  Combination therapy  3 drugs from 2 or more classes prey smildens  Fixed-dose combination – tenofovir (TDF), lamivudine (3TC), & dolutegravir (DTG/TLD) NRTI NRTI INSTI doubleDTGdose byRIF Adverse Effects Renal failure, reduced bone density, NRTIs Tenofovir & lamivudine anaemia NNRTIs Efavirenz & nevirapine CNS disturbances, rash, liver toxicity GI upset, dyslipidaemia, hepatitis, & PIs Lopinavir & darunavir rash Boostedw ritonari Mild CNS side effects, insomnia, rise INSTIs dolutegravir in serum creatinine Drug Interactions Cytochrome P450  This pathway metabolises some ARTs Boosting PIs with Ritonavir  Increases drug plasma levels  Allows for less frequent dosing  Reduces resistance risk Rifampicin (TB drug)  Reduces DTG & PIs levels  To counteract – double DTG/Lopinavir doses Monitoring Patients On ART  Primary marker Viral Load  Viral suppression should be achieved within 6 months  Regular monitoring to detect treatment failure early on Adherence  Over 90% adherence is required to prevent resistance Toxicity Monitoring  Regular assessment for side effects Mechanisms Of Action & Resistance Of ART MOA Nucleoside Reverse  Chain terminators Transcriptase Inhibitors  Prevents addition of further nucleotides (NRTIs) during reverse transcription 2ⁿᵈstep Non-Nucleoside Reverse  Bind to and alter reverse transcriptase Transcriptase Inhibitors enzyme (NNRTIs)  Inhibits its function Protease Inhibitors  Prevent viral protease from cleaving (PIs) polyproteins Gag a Pol  Halting maturation of new virions Integrase Strand Transfer  Inhibits integration of viral DNA with host Inhibitors (INSTIs) genome  Entry Inhibitors  Blocks virus from entering host cells  Targets gp120 or CCR5 co-receptor MOR  Develops through mutations in viral proteins targeted by ARTs  Reduce binding affinity of drug or alter enzyme’s active side Nucleoside Reverse Discrimination Pathway: Transcriptase Inhibitors  Reverse transcriptase preferentially (NRTIs) incorporated natural nucleotides over NRTIs  E.g. M184V mutation for Lamivudine resistance Excision Pathway:  NRTIs removed from DNA chain after incorporation  E.g. TAM mutations for Zidovudine resistance Non-Nucleoside Reverse  Single NNRTI-binding pocket mutation Transcriptase Inhibitors  High-level resistance (NNRTIs)  E.g. K103N mutation for Efavirenz & Nevirapine resistance Protease Inhibitors  Mutations in substrate-binding domain (PIs)  Reduce binding affinity between PIs and viral protease enzyme HIV Drug Resistance Testing Indications  Virological Failure – 2 VL > 1000 copies/ml after 2 years on ART despite good adherence  Baseline resistance testing – perinatally infected infants whose mothers failed on ART & individuals who failed PrEP infants Baseline or testing EhmtYeah ThosewhofailedonPrep  mutations Genotyping specific Testing Methods in the presence on phenotyping ability of a virus grow to Genotyping Resistance  Detects specific mutations in viral genes Testing  E.g. reverse transcriptase or protease genes  Sequencing  Cost-effective & short time Phenotypic Resistance  Measures ability of the virus to grow in Testing presence of ART  More expensive termingrity populations or 997in Considerations  Test done while patient is still on ARTs  Min VL of 1000 copies/ml required for accurate results  Genotypic testing – can’t detect minority populations of resistance or those in latent reservoirs Staying Updated With ART Guidelines  2023 SA ART guidelines recommend Dolutegravir-based ART o Due to its high genetic barrier to resistance, tolerability, & availability in fixed-dose combinations  New drug classes – capsid inhibitors (Lenacapavir) & long-acting injectables (Cabotegravir) ANTIVIRALS Principles Of Antiviral Therapy Introduction  Viral infection  significant morbidity and mortality  Antiviral development achieved by targeting viral enzymes or proteins essential for viral replication Key Principles  Inhibit viral functions without damaging host Selective Toxicity  Target viral-specific enzymes or processes  Most effective early in course of infection Timing  Most infections are acute  Delayed treatment limits clinical benefits  Prevents resistance Combination Therapy  Especially in HIV  Prophylaxis Preventive Use  Immunocompromised individuals  Those exposed to high-risk infections EgPrEPHIV Common Antivirals Used Nucleoside/Nucleotide Analogues mimicnucleotides chaintermination  Herpes Simplex Virus (HSV) Acyclovir, Valacyclovir thymidinekinase  Varicella-zoster virus (VZM) Ganciclovir, Valganciclovir  Cytomegalovirus (CMV)  HIV Tenofovir  Chronic Hepatitis B MOA  Inhibits viral proteases  Mimic nucleotides used by viral polymerase to make DNA/RNA  Cause lack of necessary 3’-hydroxyl group  chain termination  E.g. Acyclovir – activated by viral thymidine kinase  inhibits viral DNA polymerase  chain termination Protease Inhibitors protease immature noninfectiousvirus Lopinavir, Darunavir  HIV Hiv1protease Glecaprevir  Hepatitis C virus (HCV) MOA  Block cleavage of polyproteins into functional viral proteins  Essential for assembly of mature virions  Virus remains immature & non-infectious  E.g. Darunavir  inhibits HIV-1 protease  prevents Gag & Gag-Pol polyprotein  production of non-infectious particles Entry & Fusion Inhibitors  CCR5 receptor antagonist for Maraviroc HIV-1 Entry  Fusion inhibitor Enfuvirtide Fusion  HIV MOA  Block virus from entering/fusing with host cell  Blocks interactions between viral envelope glycoproteins & co-receptors  E.g. Enfuvirtide  binds gp41 of HIV-1 envelope glycoprotein  prevents conformational changes needed for membrane fusion Neuraminidase Inhibitors Oseltamivir, Zanamivir  Influenza MOA  Inhibit neuraminidase  prevents release of new viral particles  stops spread  Traps newly formed virions in host cell  Prevents spread NS5A Inhibitors Daclatasvir, Ledipasvir  HCV MOA  Target NS5A protein (critical for replication & assembly)  HCV  Inhibits both viral replication & assembly  E.g. Ledipasvir  binds to NS5A Other  COVID-19 Paxlovid (nirmatrelvir-ritonavir)  Inhibits viral protease Application Herpes Simplex Virus (HSV) & Varicella Zoster Virus (VZV)  Acyclovir/valacyclovir  Inhibit viral DNA synthesis  Reduce symptom severity & viral shedding Cytomegalovirus (CMV)  Ganciclovir/valganciclovir  Especially in immunocompromised  Chain terminators HIV  Combination ART  Nucleoside analogues (tenofovir) and protease inhibitors (darunavir)  Reduces chance of resistance  Ensure suppression of viral replication  Maraviroc – HIV-1 that sues CCR5 co-receptors  Prevents viral entry into CD4+ T cells Influenza  Oseltamivir/zanamivir  Neuraminidase inhibitors  Most effective when given within 48 hours of symptoms onset  Chronic Hepatitis C (HCV)  Sofosbuvir + ledipasvir  Inhibits viral replication and assembly  Highly effective  Cure if given for 8-12 weeks COVID-19  Paxlovid (nirmatrelvir-ritonavir)  Inhibits SARS-CoV-2 protease  Reduces viral replication and symptom inhibitors Entry Fusion interaction ofviralglycoproteins corec Nucleotide NucleosideAnalogues Mimicnucleotides chaintermination 9 11 deavage apolyprotein maraviroccorsrecantagonistchl lovir Acyclovir.valacyclovir.gancic piety gain.it itgigeapiigvir Enfuvirtide bindsgpal conformation valganciclovirtenofovir changesforfusion Anti Virals Neuraminidaseinhibitors Earticies spread itntEippiapi replication assembly EE niirotaneanti VIRAL VACCINES Immunological Principle Of Underlying Vaccination  Used immune system’s memory  Based on body’s ability to recognise antigens from pathogens & induce an immune response  1st exposure to antigen  antibodies and memory cells  Re-exposed  immune system responds quickly and robustly Adaptiveimmunity Primary Immune During 1st encounter with antigen. Takes days to Response develop. Secondary Immune On re-exposure, memory cells respond immediately, Response neutralising the pathogen before it causes dx. What Is A Vaccine  Biological preparation that stimulates the immune system to produce go a antibodies and memory cells. Tcell dependent cellmediated  Weakened (attenuated) or killed forms of a pathogen  Or it contains specific proteins/genetic material from the virus Vaccination Process of administering a vaccine. Process by which a person becomes protected from a Immunization dx through vaccination. Properties Of An Ideal Vaccine Inexpensive Widespread use Stable Easy to store and distribute Easy to administer Min discomfort and for all age groups Effective Long-lasting protection Safe Min side effects & can be used for immunocompromised Mucosal & systemic Targets all entry points immunity Humoral and cell- Comprehensive immune response mediated immunity Cross-reactive Protection against multiple strains or subtypes Benefits Of Vaccination Herd Immunity  Vaccination of large portion of population  Interrupts transmission  Protects the unvaccinated Reduction In Dx Cases  Lower incidence of communicable dx Decrease In Hospitalisation And Deaths  Reduced burden on healthcare system  Prevents severe dx Eradication/Elimination  Eliminate dx – small pox  Drastically reduce occurrence – polio close to eradication Limitations Of Vaccines  Delay response – immunity takes 2 weeks post-vaccination  Not 100% effective – effectiveness varies from person-person  Booster doses  Maternal antibodies – may interfere with infants  reduced efficacy  Immunocompromised patients – less effective or contraindicated (live vaccines)  Short-term protection – some only protect for a limited amount of years Side Effects Of Vaccines Common Side Effects Rare Side Effects  Mild & usually resolve quickly  Seizures  Local inflammation  High fever  Pain  Rash  Redness at injection side  Anaphylaxis  Mild fever  Irritability Specific Risks By Vaccine Type Risk of incomplete inactivation leading to immune- Inactivated Vaccine mediated disease. Risk of reversion to virulence or causing dx in Live Attenuated Vaccine immunocompromised. Elimination Vs Eradication Of A Virus Reduction of dx in specific geographic area to near Elimination zero, but continued vaccination and surveillance prevents re-introduction. E.g. polio. Complete and permanent worldwide reduction of a dx Eradication to zero cases, making continued vaccination unnecessary. E.g. smallpox. VIRAL HAEMORRHAGIC FEVER Epidemiology  Viral family – Bunyavirales, Filoviridae, Flaviviridae, Arenaviridae  Zoonotic – transmitted from animals  humans Geographic Distribution Restricted to areas where their natural reservoirs and vectors are endemic:  Ebola & Marburg – Sub-Saharan Africa inclSA  Crimean-Congo Haemorrhagic Fever (CCHF) – Africa, Balkans, and Middle East  Rift Valley Fever – Sub-Saharan Africa – outbreaks in livestock  Lassa Fever – West Africa Transmission  Ebola & CCHF – human to human  Direct contact with bodily fluids  Strict infection control needed  SA – CCHF most common Management Of A Suspected Case Clinical Approach History  Recent travel to endemic areas  Contact with livestock, animals, or patients with known VHF  Occupational exposure – farmers, abattoir workers, vets  Arthropod bites or tic exposure Examination  Early – fever, headache, myalgia common in VHF  pthrombocytopenia Bleeding – petechia, epistaxis, haematemesis, melaena  Multi-organ involvement – jaundice, altered mental state, shock, multi-organ failure Infection Control  Isolate patient and wear full PPE o Full PPE – double gloves, double gowns, visors, masks, shoe coverings  Notify authorities – category A notifiable dx – report within 24hrs  Supportive care – fluid/electrolytes, blood transfusions  Disinfection – chlorine-based disinfectants Laboratory Testing FBCa coagulationstudies Haematology LFTs Diagnostic Approach Biochemistry Electrolytes Virology PCR for VHFviruses Routine Tests Conducted at National Health Laboratory Service (NHLS) labs for:  Haematology – FBC, coagulation studies (thrombocytopenia common in VHF)   Biochemistry – electrolyte and liver function tests  Virology – PCR for VHF viruses NICD Testing  Viral antigen detection or isolation  Biosafety level 4 labs  Available at National Institute for Communicable Diseases (NICD) Specimen Collection  Extreme care taken  Triple packaging  Outer package – labelled with biohazard warning Common VHFs In SA Crimean-Congo Haemorrhagic Fever (CCHF) Reservoir  Hyalomma ticks  Humans infected through tick bites or contact with infected animal tissue Transmission  Human-to-human  Blood or bodily fluids Incubation  3-7 days musclepain p  Followed by fever, headache, myalgia  Haemorrhage in some cases Mortality  3-30% Diagnosis  PCR  IgM & IgG – by day 5 Management  Supportive therapy  Antiviral – ribavirin Ebola Virus Disease (EVD) Reservoir  Bats   Transmitted via direct contact with infected animals or humans Geography  Central & west Africa  Guinea, Sierra, Leone, Liberia Mortality  50-90% Transmission  Direct contact with blood or bodily fluuids Management  Supportive care – fluid management and ICU  Monoclonal antibody therapy – Inmazeb, Ebanga Vaccine  Ervebo for Zaire ebolavirus VIRAL INFECTIONS IN IMMUNOCOMPROMISED PATIENTS Definitions DiGeorge Syndrome T cells Primary Immunodeficiency B cells Selective IgAdeficiency  Genetic or congenital  Immune system defect is intrinsic to immune cells  E.g. DiGeorge Syndrome – T-cell defects  E.g. Selective IgA deficiency – B-cell defects since IgA is an antibody  Diagnosed in infancy or childhood  Majority X-linked, affecting males Secondary Immunodeficiency  Acquired conditions posttransplant peg  Caused by external factors – infections (HIV), malignancy, immunosuppressive therapy, or environmental factors  More common  Diabetes or treatments (chemo) can impair immune function  increased susceptibility to infections Clinical Features, Diagnosis, And Management  Atypical presentations, more severe dx, & poor response to antivirals  Shed viruses for longer, be highly infectious, & poor response to vaccines  Diagnosis – PCR or biopsy o Serological tests unreliable due to inadequate antibody repones  Management – antiviral therapy tailed to severity of infection and adjustments in immunosuppressive therapy HIV HSV 2 a vav 2 Groups Of Immunocompromised Transplant HIV-Infected Patients  Progressive impairment of immune function  Vulnerable to opportunistic infections  Advanced HIV (CD4 < 50 cells/µL)  severe conditions (CMV retinitis & PML) Common co-infections :  HSV-2  increased risk of HIV transmission HerpesSimplex 2  VZV  recurrent shingles VaricellaZoster Virus Treatment:  ART – immune restoration  Specific antivirals – acyclovir for HSV Transplant Recipients  Immunosuppressive therapy for organ rejection  Increases infections (CMV)  reactivate post-transplant Monitoring a preemptive antivirals Risk Periods:  Pre-engraftment – up to 30 days  Early post-engraftment – 30-100 days  Late post-engraftment - > 100 days Cytomegalovirus (CMV):  Significant cause of morbidity and mortality  Monitoring & pre-emptive antiviral therapy reduces burden Other Infections:  BK Virus  nephropathy in renal transplants  JC Virus  PML in haematopoietic stem cell transplants Influenza Immunisation In Immunocompromised 19Eivated polio  Less effective (especially if CD4 > 200 cells/µL)  Live vaccines contraindicated due to risk of vaccine strain proliferation  Transplant recipients – immunization should be done before transplants and re-vaccination post-transplant  Recommended vaccines – influenza (annual), hepatitis B (if non-immune_, and inactivated polio post-transplant  EMERGING & RE-EMERGING VIRAL INFECTIONS Understanding The Concept Of Emerging And Re-Emerging Newly discovered or previously known viruses that Emerging Viruses are increasing in incidence or geographic Majorityarezoonotic distribution. Viruses that were under control but are now Re-Emerging Viruses resurging. Due to change of host, environment, or mutation. Infections transmitted from animals to humans or Zoonoses person to person. Factors That Drive Emergence Viral Factors Preckmanitations newstrains Host change to able infect humans High Mutation Rate:  RNA viruses due to lack of proofreading during replication  E.g. avian influenza, Nipah, & coronaviruses Genetic Recombination And Re-Assortment  New viral strains that may infect new hosts  E.g. influenza Change Of Host Range Or Tropism  Cross species barriers  Become able to infect humans Human Factors  Population growth, urbanisation, and overcrowding  Global travel and trade  Changes in agricultural practices, deforestation, and live animal markets   Human behaviour and practices o Wildlife hunting, live animal trade, and handing of animals Ecological And Environmental Factors  Climate change and ecological disruption o Proliferation of vectors (mosquitoes) and pathogens  Biodiversity loss and deforestation Epidemiology  Epidemic and pandemic o E.g. SARS, MERS, COVID-19, Ebola & Zika Public Health Impact  Strains health system  Increases mortality  Economic loses  Disrupts societal functions Zoonotic Viruses  Majority of emerging viruses  Significant potential for rapid transmission and global spread  Due to human behaviours and environmental changes Notable Emerging Viral Infections And Clinical Syndromes They Cause SARS-CoV-2 (COVID-19)  Global pandemic in 2019  Transmission – respiratory droplets  Symptoms – fever, cough, difficulty breathing, loss of taste and smell  Prevention – vaccination, social distancing, hygiene practices Ebola Virus  Viral haemorrhagic fever  High fatality   Transmission – contact with bodily fluids of infected person or animals (bats/primates) Zika Virus  Flavivirus  Transmission – mosquitoes  Symptoms – fever, rash, conjunctivitis, joint pain  Pregnant women – congenital abnormalities like microencephaly Nipah Virus  Transmission – zoonotic – bats  Symptoms – encephalitis and severe respiratory illness  Prevention – limiting human-bat interactions MERS-CoV (Middle East Respiratory Syndrome)  Transmission – contact with camels  Severe respiratory illness and high mortality Prevention And Prediction Strategies Prevention One Health Approach  Recognises connection between humans, animals, and environmental health. Surveillance Monitors:  Animal populations  Human cases  Vectors for early detection Vaccination And Vector  Prevents outbreaks Control  Influenza, tallow fever, dengue Public Health Education  Targets high-risk populations  Food handles, hunters, healthcare workers Outbreak Response  Strengthening healthcare infrastructure  Adherence to infection prevention and control (IPC)   Minimise nosocomial spread Prediction Geospatial Models  Hot spots of emergence  Regions with high human-animal- environmental interactions Phylodynamics  Combines epidemiological and molecular data  To understand evolution and spread Surveillance Of Animal  Cataloguing & sequencing animal viruses Viruses  Early warnings of zoonoses with potential to infect humans  E.g. Bombali virsues in bats VIRAL INFECTIONS OF SKIN & MUCOSA Overview  Specific – herpes zoster’s dermatomal vesicles  General – urticaria hives usuallydue to anallergicreaction  Diagnosis – history, clinical presentation, epidemiology  Pathogenesis – direct viral replication in skin (HSV/HPV) or systemic spread (measles/rubella) Dermis Pathophysiology Direct Inoculation  HPV & HSV  Replicate within dermis Systemic Spread  Measles & rubella  Systemic viremia  skin & mucosal manifestations Routes of Infection  Direct contact   Respiratory droplets  Systemic spread from internal focus Diagnosis Clinical Diagnosis  Some conditions can be identified in clinical features  E.g. chickenpox, herpes zoster, molluscum contagiosum Laboratory Conformation  PCR testing – gold standard  Severe cases or atypical presentation Common Viral Infections Of Skin And Mucosa Measles Transmission  Highly contagious  Respiratory droplets Symptoms  Fever, cough  Conjunctivitis  Coryza  Koplik’s spots (buccal mucosa)  Erythematous maculopapular rash (starts on face) Complications  Pneumonia  Diarrhoea  Neurological issues – encephalitis Diagnosis & Management  Notify authorities  Confirm with PCR or IgM testing  Supportive care – hydration & vit A Rubella (German Measles) Lesssevere infectiousthanmeasles Transmission  Respiratory droplets  Clinical Features  On-confluent maculopapular rash starting on face  Lymphadenopathy  Pregnancy – congenital rubella syndrome Prevention  Rubella vaccine in childhood Parvovirus B19 (Erythema Infectiosum) Symptoms  “slapped cheek” rash  Maculopapular rash on trunk Complications  Arthralgia in adults Pathogenesis  Immune complexes  rash Human Herpesvirus 6 (Roseola Infantum) Transmission  Saliva Clinical Features  High fever  Maculopapular rash  Common in infants Complications  Febrile seizures dueto To 38 C  Encephalitis (rare) Herpes Simplex Virus (HSV) Types  HSV-1 (oral) Encephalitis  HSV-2 (genital) Meningitis Manifestations  Vesicular lesions  Gingovostomatitis  Hepatic whitlow lesiononfinger  Encephalitis – HSV-1  Meningitis – HSV-2 Management  Acyclovir  Symptom management Varicella-Zoster Virus (VZV) Primary Infection  Fever (Chickenpox)  Pruritic vesicular rash  Infectious until lesions crust Reactive (Herpes Zoster)  Painful dermatomal rash  Complications – postherpetic neuralgia Management  Chickenpox – supportive care  Antiviral treatment for high-risk or complicated cases Enteroviruses Diseases  Hand, foot, & mouth  vesicular lesions  Herpangina Diagnosis  Clinical assessment Epstein-Barr Virus (EBV) Clinical Presentation  Infectious mononucleosis  Fever  Pharyngitis  Lymphadenopathy  Rash (sometimes) Complications  Rash when amipicillin/amoxicillin is given Human Papillomavirus (HPV) Types  Cutaneous – warts  Mucosal – genital warts & cervical neoplasia Management  Lesion removal & vaccine (prevention) Molluscum Contagiosum Transmission  Direct contact   Children Clinical Features  Umbilicated, pearly nodules  Last up to months Management  Supportive Therapy – hydration, antipyretics, and wound care  Antiviral Meds – severe cases or high-risk populations (e.g. immunocompromised)  Vaccination – preventing measles, rubella, varicella, and HPV-related conditions