Respiratory Tract Infections PDF Lecture Notes

🦠 Respiratory Tract Infections Module Microbiology Date @October 3, 2024 Lecturer Dr Ryan Kean Week Week 2 symptoms of patient, clinical scenario, what samples to tak...

🦠 Respiratory Tract Infections Module Microbiology Date @October 3, 2024 Lecturer Dr Ryan Kean Week Week 2 symptoms of patient, clinical scenario, what samples to take, how to treat it Learning Outcomes Respiratory pathogen transmission and host defence mechanisms Infections of the upper respiratory tract Streptococcus pyogenes Infections of the lower respiratory tract Streptococcus pneumoniae Infections of the immunocompromised COPD Post identification processes Antimicrobial Susceptibility Testing (AST) Pathogens of the upper and lower respiratory tract Following list is not exhaustive, just a short list of what viruses / bacteria / fungal pathogens cause infections of the respiratory tract. Respiratory Tract Infections 1  Bacterial Top 3 we focus on in this lecture Streptococcus pneumoniae Streptoccous pyogenes Haemophillus influenzae Staphylococcucs aureus Corynebacterium diptheriae Mycobacterium tuberculosis Nontuberculous mycobacteria Pseudomonas aeruginosa Moraxella catarrhalis Stenotrophomonas maltophilia Bordetella pertussis Viral Influenza Parainfluenza Adenovirus Rhinovirus Respiratory syncytial virus Fungal Aspergillus spp. Cryptococcus neoformans/gattii Histoplasma capsulatum Paracoccidioides brasiliensis Respiratory Tract Infections 2  Blastomyces dermatitidis Host defence mechanisms Physical Estimates suggest that the average human can inhale over 500,000 organisms in a single day. Why do we not succumb to infection more often? Physical defences are the first line of defence Saliva - constant flow and different enzymes to eliminate threats (peroxidase, lysozymes, lactoferrin - an iron scavenging molecule which binds free iron circulating in bodily fluids). Hairs in anterior nares - act as a physical barrier Cough and epiglottic reflex - get rid of potential pathogens Ciliated cells in the respiratory tract - lined with mucus which traps microbial threats Immunological If physical mechanisms fail to remove some microbes, then humoral and cellular defences are also important Adenoids and tonsils Lymphoid tissues Alveolar lining fluid Specific and non-specific antimicrobial peptides Alveolar macrophages Humoral and cell-mediated immunity Cytokines Antibodies Polymorphonuclear leukocytes and monocytes Respiratory Tract Infections 3  Infections are transmitted through particles / aerosols which are termed classified by size of particle and distance able to travel. Saliva and nasal secretions also transmit infections. Fomites are any sort of inert substance which a pathogen can fall on / colonise on acting as intermediate of the infection. There are many factors which can affect transmission in the environment. Laboratory diagnosis Respiratory Tract Infections 4  Assess clinical history and suitability of specimen Collect appropriate sample to culture specimen Interpretation of significance of culture Isolate any pathogen(s) Identify pathogen(s) Antimicrobial susceptibility testing (AST) - is very important as antimicrobial resistance is a big issue. Release report with significance of pathogen and sensitivities or as NG/NSG. Respiratory Tract Infections 5  Laboratory methods are based on HPA Standards for Microbiology Investigations (SMI) Types of sample Key things to consider are where the patient is showing signs of infection and also the status of a patient (don't use invasive techniques if the patient is very sick). Sputum sample has to be digested before analysed. BAL - bronchial alveolar lavage Culture conditions A number of different techniques are used to correctly identify the causative pathogen(s). Both selective and non selective agars are used. Differential agar and enrichment broths are used to encourage growth are also used. Interpretation of isolated organism can be complicated Further testing should be performed to identify the pathogen once the organism is grown: - Gram stain - Growth requirements - Biochemical testing Respiratory Tract Infections 6  Infections of the upper respiratory tract Bacterial URTI Common URTI infections which are associated with bacteria: Pharyngitis/tonsillitis – infection of the tonsils and tissues at the back of the throat. Rhinitis – inflammation of the nasal cavity. Sinusitis – infection of the sinuses. Laryngitis – infection of the larynx (voice box). Otitis media – inner ear canal infection. ~ Sinusitis ~ Respiratory Tract Infections 7  Infection of the sinuses, typical symptoms include: Fluid build up in the air filled pockets in the face Fluid allows microbes to grow, typically viruses but also bacteria Runny and stuffy nose are the generic signs and symptoms. Facial pain is common with sinusitis due to build up of pressure and also headaches. Sore throat, cough and bad breath. Symptoms usually last up to 10 days. Treatment is usually self care of pain relief, antibiotics rarely given. Steroid nasal sprays for more severe cases. ~ Laryngitis ~ Infection of the larynx, typical symptoms include: irritation and inflammation of the larynx Hoarse or croaky voice, occasional losing of the voice Sore throat Respiratory Tract Infections 8  Irritating and consistent cough Symptoms usually last between 1-2 weeks with the initial onset of infection the worst Treatment is usually self care of pain relief/solutions to gargle for throat pain. Antibiotics rarely given. ~ Otitis Media ~ Infection of the inner ear, typical symptoms include: Blocking of the Eustachian tubes which results in fluid build up Ear pain and trouble hearing Drainage of fluid from the ear Headache and sometimes fever Symptoms and signs of infection is usually rapid. Treatment is usually self care and pain relief as infection usually self limiting. Antibiotics rarely given, used in more severe/reoccurring cases. Respiratory Tract Infections 9  ~ Pharyngitis ~ Inflammatory syndrome of the pharynx. Generally caused by viruses – common cold (Adenoviruses). Most frequent bacterial aetiology – Streptococcus pyogenes: Adults 10-15% cases Children 15-30% cases Other bacterial causes are generally negligible Streptococcus Pyogenes Respiratory Tract Infections 10  Non-motile Gram-positive cocci, arranged in chains Fastidious facultative anaerobe Also known as β-haemolytic, Lancefield group A (aka class A strep) Carriage rate of 15-20% Susceptible to most antibiotics Penicillin generally used for GAS pharyngitis Stain purple Erythromycin would be next antibiotic of choice. Lancefield Groupings Pioneering findings from Rebecca Lancefield in 1933 Her technique is based upon serological analysis of specific carbohydrate (C) antigens within the cell wall of β haemolytic Streptococci. These are arranged in 20 groups: A-H and K-V Most relevant - A, B, C, D, F and G The test uses the principle of antigen (cell wall carbohydrate) -antibody binding. Respiratory Tract Infections 11  ASSAY PRINCIPLE A number of different commercial kits are available, which are rapid, cheap and work at room temp. Within the kit extraction reagents 1 &2 extracts the specific C antigen from the cell wall using a nitrous acid extraction. Extraction reagent 3 contains a neutralising reagent. Extract (antigen) can the agglutinate with latex beads conjugated with specific rabbit antibodies. S. pyogenes virulence factors Remember stuff highlighted in red. Respiratory Tract Infections 12  SIC – Streptococcal inhibitor of complement. Interferes with the formation of MAC within the complement cascade (inhibits the very end stage of the classical complement pathway). Undergoes antigenic variation to evade the immune response. Superantigens: at least 11 recognised in S. pyogenes. Exotoxin which is associated with scarlet fever and toxic shock like syndrome. Cell wall components LTA and peptidoglycan also trigger an inflammatory response. Capsule – antiphagocytic function so helps the organism avoid the immune response. Non-antigenic because of similarities to host cell connective tissue. Also prevents organisms being efficiently phagocytosed. M protein – the major virulence factor of GAS. It has antiphagocytic and anti- complement properties. They are cytotoxic to neutrophils. They are able to bind the Fc region of an antibody and as interfere with the factor H interaction in the C3b binding of the complement cascade. More than 80 of these proteins have been identified highlighting antigenic variation. General rule of thumb is that the lover numbered M protein types are rheumatogenic and can lead to autoimmune Respiratory Tract Infections 13  rheumatic carditis. Also the weakest virulence factor because plasma B cells generate antibodies against M protein, rendering it susceptible to macrophages Large variety of extracellular enzymes: Streptolysin O – oxygen liable (inactivated by oxygen). Destroys red and white blood cells and is the reason for the beta haemolytic appearance on agar. It is antigenic Streptolysin S – oxygen stabile. Also responsible for beta haemolysis but is not antigenic Dnase – degrades DNA Proteases: SpyCEP – surface expressed protease which catalyses the cleavage of the neutrophil chemokine IL-8. Streptokinase – activates the proteolytic enzyme plasmin which breaks up finbrin in blood clots. Pili, FBP, LTA and M protein – all have roles in the adherence to host cells. Hyaluronic acid capsule - also a component of host tissue to body cannot differentiate between self and non self. Respiratory Tract Infections 14  There are a number of things which affect immune response: Opsonin – molecule which enhances phagocytosis. NET – neutrophil extracellular trap. These are secreted by neutrophils which can trap bacteria. Made of DNA so S. pyogenes DNase is able to degrade these structures. Post pharyngitis complications Scarlet fever: Often presents alongside pharyngitis. Rash begins on trunk and spreads to extremities Can also affect the tongue – ‘strawberry tongue’ Typically lasts around one week Often self limiting but can be treated with penicillin, cephalosporin / macrolide can be used in case of allergy There are a number of post pharyngitis complications which can occur. These are extremely rare but have potentially life threatening consequences. Generally will affect children between 5-15, and will occur after an untreated infection. Introduction of penicillin means consequences are very rare. Respiratory Tract Infections 15  Complications are as a result of a delayed antibody mediated response which results in an autoimmune mechanism known as molecular mimicry. This occurs when similarities between foreign (in this case M protein of S. pyogenes) and self antigens (M protein is very similar to myosin found in heart valves) and this can result in the immune system not being able to be distinguish between self and non self. As it cannot distinguish between these, this results in the generation and activation of cross reactive T and B cells. This can result in an autoimmune response in various tissues and organs of the body, these include myocarditis of the heart muscle, Sydenham's Chorea, this affects the brain and can result in various neurological problems. It can cause subcutaneous nodule and a rash called erythema marginatum and finally can cause the multi system disorder called rheumatic fever. Heart Myocarditis – inflammation of the heart muscle Brain Sydenham’s chorea - Neurological disorder leading to uncontrolled, erratic movements Rheumatic fever Multi-system inflammatory disorder involving the heart, joints, and skin Skin Subcutaneous nodules and a rash called erythema marginatum PATHOGENESIS : PHARYNGITIS → RHEUMATIC HEART DISEASE ~dont need to memorise the following it is just for info Respiratory Tract Infections 16  A. GAS infection results in immune system stimulation B. Antigen presenting cells processing and presenting GAS antigens stimulate T and B cell immunological responses C. GAS and self cross-reactive activated T cells enter the lymphatics system, through the heart and lungs and to the mitral valve → inflammation (F) D. GAS and self cross-reactive activated antibodies also enter the lymphatics system, through the heart and lungs and to the mitral valve → inflammation (F) E. Breaching of mucosal barrier by GAS bacteria/components and deposition on mitral valve → inflammation (F) F. *inflammation* G. Mitral valvulitis with granulomas Infections of the lower respiratory tract Respiratory Tract Infections 17  Pneumonia PNEUMONIA CAN BE SPLIT INTO 2 DIFF SUBTYPES; COMMUNITY ACQUIRED AND NOSACOMICALLY AQUIRED PNEUMONIA (acquired 48hrs after hospitalisation) THE STATS For a long time was in the top 10 of most common cause of death in all age groups in US Recently dropped out in 2021 (12th most common) Leading cause of death in the 65-years or over group Single most common cause of infection-related mortality UK stats 220,000 people diagnosed annually More than 25,000 deaths The leading cause of death in children worldwide Kills an estimated 1 million children every year 2500 per day Respiratory Tract Infections 18  Vaccination has reduced the figures by 47% (2000-2015) (Unicef, 2016). Can be treated with antibiotics, but less than 29% of children with pneumonia receive the antibiotics they need (WHO, 2012). COMMONLY ACQUIRED PNEUMONIA (CAP) Incidence: 1 in 100 people per year 20-40% hospital admission rate Typically affects age range between mid 50s and beyond Occurs all year but peaks in mid-winter and early spring Most patients (up to 90%) have underlying medical conditions COPD, diabetes, CVD Mortality rate 5-15%, around 30% in those admitted to ICU HOSPITAL ACQUIRED PNEUMONIA (HAP) Respiratory infection which develops more than 48 hours after hospital admission Second most common type of nosocomial infection Mechanical ventilation is a major risk factor - VAP (ventilation associated pneumonia) Increased risk of infection by the presence of underlying disease and by various interventions and procedures. CLINICAL CONSIDERATIONS AND CHALLENGES Multifactorial nature of causative agents makes it difficult to diagnose pneumonia Both clinical and aetiological diagnosis becomes difficult No single empiric antimicrobial course can cover all of the potential causes Respiratory Tract Infections 19  Challenges are becoming more complicated with the emergence of AMR Typical pathogens: Streptococcus pneumoniae Haemophillus influenza Staphylococcus aureus Moraxella catarrhalis Aypical pathogens: Legionella spp (outbreaks) Mycoplasma pneumoniae Chlamydophilia pneumoniae Gram negative pathogens: Pseudomonas aeruginosa Klebsiella pneumoniae STREPTOCOCCUS PNEUMONIAE Gram-positive diplococcus, alpha-haemolysis on blood agar, polysaccharide capsule Does not have Lancefield antigens Carriage rate highest in first two years then declines Infections of the respiratory tract include otitis media, sinusitis, pneumonia Respiratory Tract Infections 20  Meningitis and bacteraemia S. PNEUMONIAE VIRULENCE FACTORS Respiratory Tract Infections 21  Its capsule makes it a successful pathogen. However, it is highly immunogenic so it is a great target for a vaccine. S. PNEUMONIAE CAPSULE Unencapsulated strains are usually avirulent Of the approx. 90 serotypes – 23 are associated with Pneumonia Prevents mechanical removal from phagocytes Masks PAMP to which complement proteins or antibodies bind Binds to factor H to degrade complement protein C3b Target of vaccines Pneumococcal Conjugate Vaccine: Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F More common in young children Does not provide protection against other serotypes Pneumococcal Polysaccharide Vaccine: All 23 serotypes People over 65 years old or long term medical conditions Respiratory Tract Infections 22  Not very effective in children younger than 2 Between 50-70% efficacy KNOW YOUR STREPS Enterococcus used to be called group D. Respiratory tract infections of the compromised host Organ transplantation, cystic fibrosis, COPD, diabetes, HIV COPD Characterised by airway inflammation and decrease in lung function 3rd leading cause of death worldwide Respiratory Tract Infections 23  Exceedingly prevalent in Western Europe especially the UK and Ireland 4-10% of European population have COPD Exacerbations significantly increase the rate of lung deterioration 50% of exacerbations caused by bacteria Haemophilus influenzae Aerobic, gram-negative coccobacillus Common commensal of the nasopharynx and throat but common pathogen of the lungs 37°C, CO2 enriched atmosphere – chocolate agar Specific growth requirements Cannot grow on unsupplemented blood agar Requires factor X (haemin) Requires factor V (NAD) H. influenzae requires both factors Respiratory Tract Infections 24  Colonises the URT Non-encapsulated 25-80% carriage Capsulated 5-10% carriage Cause both invasive and non-invasive infections Some strains have a polysaccharide capsule Serotypes a-f, B is the most common Non-encapsulated strains referred to as ‘Nontypeable Haemophillus influenza’ Respiratory Tract Infections 25  One of the WHO 12 priority pathogens in 2017 Linked to resistance to the beta lactam antibiotic ampicillin Group B is most commonly associated with invasive disease such as meningitis Hib vaccine available – introduced in 1992 Vaccination has decreased infection rate by 90% Moraxella Catarrhalis Aerobic, gram-negative diplococcus Common commensal of the upper respiratory tract and causes a variety of infections 37°C, aerobic– blood and chocolate agar Most strains are βlactamase positive Incidence thought to be underappreciated in COPD Antimicrobial Susceptibility Testing (AST) Microbiological techniques where a pure culture of a single microbe is grown in the presence of an antimicrobial agent Respiratory Tract Infections 26  AST can provide a direct analysis of the susceptibility of a microbe to an antimicrobial at specific concentrations – in vitro Some methods allow for quantitative analysis which can define the MIC of the antimicrobial MIC – minimum inhibitory concentration Findings can then tailor antimicrobial prescribing to the patient Disc Diffusion One of the originally used AST methods - Method Analysis based on the zone of inhibition (ZOI) of growth on solid agar ZOI – the area around where the antimicrobial has been positioned (usually a disc). The radius of the ZOI correlates with the susceptibility of the organism to the antimicrobial Laboratory automation can now dispense disc and measures ZOI automatically Automatic processes also allow for more rapid results turnaround 4, 6 and 8 hours directly from positive blood samples Respiratory Tract Infections 27  ETEST Similar principle to the disc diffusion test ETEST strip is a predefined gradient of antimicrobial concentrations and can determine the MIC Allows for testing of slow growing organisms which automated testing cannot work with Readily available for many antibiotics and antifungals Preparation tutorial - https://www.youtube.com/watch?v=K0nrFj8lFfA Broth Dilution Respiratory Tract Infections 28  96 well plate. lanes 10-10 is a serial doubling dilution of our antimicrobial. Lane 11 is positive control; no antimicrobial at all, just growth media of microbe. Lane 12 is negative control; only media no microbes. Vitek 2 It can perform susceptibility testing and identify the bacteria. Automated system which uses growth-based technology and colorimetric cards which can both identify bacteria and perform AST Integrated with LIMS Can detect >100 resistance mechanisms AST and ID - Technology Advances - Next Generation Sequencing (NGS) Respiratory Tract Infections 29 Respiratory Tract Infections 30