Brunel University London 2024 Introduction to Medical Sciences 1 PDF

Introduction to Medical Sciences 1 Viruses, fungal and parasitic infections Diagnostic tests in infectious diseases Copyright © Brunel University London v.3 2024. All rights reserved. Viruses, fungal and parasitic infections Dr William Lynn 2024 Version 3 willi...

Introduction to Medical Sciences 1 Viruses, fungal and parasitic infections Diagnostic tests in infectious diseases Copyright © Brunel University London v.3 2024. All rights reserved. Viruses, fungal and parasitic infections Dr William Lynn 2024 Version 3 [email protected] Copyright © Brunel University London v.3 2024. All rights reserved. Viruses, fungal and parasitic infections Diagnostic tests in infectious diseases Copyright © Brunel University London v.3 2024. All rights reserved. In this session We will review the tests available for the diagnosis of infectious diseases These tests complement our clinical skills and are vital in targeting appropriate therapy This will include tests used for bacteria as well as those for viruses, fungi and parasites This will illustrate the use of tests with some specific clinical examples Copyright © Brunel University London v.3 2024. All rights reserved. Why is it important to try and identify the organism? Improve patient outcome Public Health Making the correct diagnosis Regional and global surveillance Appropriate antimicrobial therapy Emergence and spread of high Best antibiotic choice risk or new pathogens Reduce risks of antimicrobial Contact tracing resistance Meningitis, tuberculosis Awareness of complications eg: Surveillance Involvement of the eye in Salmonella gastroenteritis cytomegalovirus infection in linked to a restaurant immunocompromised host Copyright © Brunel University London v.3 2024. All rights reserved.  What samples can we take easily Urine Swabs Wounds, skin, Blood throat, urethra etc Stool Pus draining from an abscess Sputum Image: Swab blood-stained pus from an infected wound. Reuben Strayer, CC BY-SA via Wikimedia Commons Copyright © Brunel University London v.3 2024. All rights reserved.  Examples of invasive samples from more complex cases Samples from deep in the lungs Via a bronchoscope Cerebrospinal fluid Lumbar puncture Bone marrow Bone marrow biopsy Deep tissue or abscess samples Image-guided aspiration or biopsy Image A: Bronchoscopy. Cancer Research UK, CC BY-SA 4.0 via Wikimedia CommonsImage Image B: Lumbar puncture. Medical gallery of Blausen. WikiJournal of Medicine 1 (2). DOI:10.15347/wjm/2014.010. ISSN 2002-4436., via Wikimedia Commons Copyright © Brunel University London v.3 2024. All rights reserved. What types of tests can we do? Microscopy Antigen detection Direct visualisation of microorganisms Identify antigens specific to organisms Culture Grow microorganisms in Nucleic acid detection laboratory Amplify and identify sequences of Immunological tests RNA or DNA specific to individual organisms Measure the immune response to specific antigens Copyright © Brunel University London v.3 2024. All rights reserved.  Light microscopy – bacteria, fungi and parasites Direct visualisation of microorganisms Unstained or stained preparations Suggested identification from the morphology and staining characteristics Sensitivity limited Need specimen containing sufficient microorganisms for detection Specificity limited Non-pathogenic organisms in samples from non-sterile body sites Rapid Not a definitive identification No antimicrobial sensitivity data Image A: Rhodamine fluorescent stain showing Mycobacteria. CDC Public Health Image Library available at www.phil.cdc Image B: Normal upper respiratory flora in Gram stained sputum smear. Ajay Kumar Chaurasiya, CC BY-SA 4.0 via Wikimedia Commons Copyright © Brunel University London v.3 2024. All rights reserved.  Electron microscopy – can visualise viruses Electron microscopes Expensive, specialised interpretation For many years only available method for rapid diagnosis No longer in routine clinical use Image A: Electron microscopy of smallpox virions CDC/ Dr. Fred Murphy; Sylvia Whitfield, Public domain, via Wikimedia Commons Image B: Electron microscopy of herpes virus. CDC/Dr. Erskine Palmer, Public domain, via Wikimedia Commons Copyright © Brunel University London v.3 2024. All rights reserved.  Immunostaining – used in specialised settings Technique that uses antibodies against specific antigens to identify pathogens in cellular samples A sample from the base of a genital ulcer has been exposed to an antibody against a herpes simplex antigen A fluorescein (FITC) conjugated immunoglobulin staining technique reveals an epithelial cell fluorescing under ultraviolet light Confirms herpes simplex virus type-2 (HSV-2) infection Image: Herpes simplex immunostaining. Dr. Craig Lyerl. CDC Public health image library. Available at www.phil.cdc.gov Copyright © Brunel University London v.3 2024. All rights reserved.  Culture – a brief overview Culture can be used on any fresh clinical specimen Added to culture media designed to encourage growth in vitro Appearance of bacterial colonies useful More detailed diagnostic tests Identify the organism based on metabolic and biochemical phenotype Anti-microbial sensitivity testing can be performed Image A: Streptococcus pneumoniae colonies (arrows) on agar. CDC public image library. Available at www.phil.cdc Image B: Blood culture bottles. Red for anaerobes, green for aerobes. James Heilman, MD, CC BY-SA 3.0 via Wikimedia Commons Copyright © Brunel University London v.3 2024. All rights reserved.  Culture – the path to antibiotic sensitivity testing Bacteria from a positive culture have been spread over agar Paper discs containing antibiotics are added and incubated at 37oC The bacteria on the left are sensitive to all of the antibiotics compared to the one on the right which is resistant to 4 and partially resistant to the one in the middle Image: Antibiotic disk sensitivity testing. Dr Graham Beards. Dr Graham Beards at en.wikipedia, CC BY-SA 4.0 via Wikimedia Commons Copyright © Brunel University London v.3 2024. All rights reserved.  Antigen detection An antigen is any molecule or Advantages particle that can be recognised by a specific Rapid and specific immunoassay antibody Easy to perform Point of care tests available Antigens may remain Not affected by antibiotics attached or may be ‘shed’ by the organism Disadvantages Looked for in clinical samples Can only detect one organism per test or to identify bacterial cultures Sensitivity less than molecular techniques No antimicrobial sensitivity data Copyright © Brunel University London v.3 2024. All rights reserved.  Antigen detection example – COVID 19 A nasal and/or pharyngeal swab is placed in fluid solubilising the viral antigens The fluid is added to the sample well and moves up the testing chamber by capillary action Antigens are captured by specific antibodies embedded in the substrate of the testing chamber floor A colour change allows rapid visualisation with control and test lines Sensitivity – 70-75% Specificity - 98-99% Image: Positive lateral flow test for COVID 19. Courtesy of Dr William Lynn. Copyright © Brunel University London v.3 2024. All rights reserved. Serological tests in infection Serological tests Measure immunoglobulin– IgM, IgG, IgA and IgE Evidence that an individual has mounted an immune response Does not necessarily indicate active infection Negative early in infection before antibody is detectable ‘Window period’ May be difficult to interpretate Cross reacting antibodies between similar organisms/species Distinguishing active infection from past infection or immunisation Poor antibody responses in severely immunocompromised individuals Copyright © Brunel University London v.3 2024. All rights reserved.  Dynamics of antibody responses Situation Pattern Acute or recent IgM positive infection IgG rising Past infection or IgM negative immunisation IgG positive Chronic infection IgM - positive or negative IgG positive Primary antibody responses. Adapted from Figure : 21.24a in Anatomy & Physiology by Queensland University of Technology. Available at https://qut.pressbooks.pub/anatomyandphysiology/chapter/chapter-21-the-lymphatic-and-immune-system/ Public domain via Creative Commons Attribution 4.0. Copyright © Brunel University London v.3 2024. All rights reserved.  Nucleic acid detection – the way forward? Direct hybridisation of RNA or DNA lacks sensitivity PCR amplification is the most common technique Practicalities Uses of nucleic acid tests Used with many biological specimens Rapid accurate diagnosis Detect multiple pathogens from Detection of antimicrobial resistance same specimen High Specificity and Sensitivity Improved clinical decision making Technology improving allowing rapid turnaround and becoming Public health more cost effective Identify new and emerging pathogens Genomic information PCR; polymerase chain reaction Copyright © Brunel University London v.3 2024. All rights reserved. Which tests are best for different types of organisms? Copyright © Brunel University London v.3 2024. All rights reserved.  Viruses Antigen detection Diagnosis of acute infection COVID 19 Microscopy and Culture Detect early infection Viruses not visible on light microscopy HIV antigen positive before antibody Viruses must be cultured inside cells Distinguish active from past infection No longer in routine use Hepatitis B Nucleic acid amplification - PCR Serology Diagnosis Diagnosis of some acute viral infections Respiratory viruses Screen for chronic infections such as HIV Dengue (travel-related) or viral hepatitis CMV in immunocompromised Confirm immunity from past infection or Monitoring the response to therapy immunisation HIV Antiviral resistance CMV, hepatitis B and HIV CMV; cytomegalovirus, HIV; Human immunodeficiency virus Copyright © Brunel University London v.3 2024. All rights reserved.  Bacteria Microscopy Antigen detection Urine, CSF, other body fluids and pus Diagnosis of some bacterial infections Positive blood culture Streptococcus pneumoniae, Limited value on sputum Legionnaires disease Culture Nucleic acid amplification - PCR Mainstay of diagnosis, identification and Not routine for most infections anti-microbial sensitivity testing Standard of care in tuberculosis Increased sensitivity compared to Serology microscopy Diagnosis of some bacterial infections Faster identification and sensitivity Syphilis testing than culture CSF; cerebrospinal fluid Copyright © Brunel University London v.3 2024. All rights reserved. Fungi Microscopy Antigen detection Able to recognise fungal elements in Fungal antigens are useful in the diagnosis clinical specimens – yeasts or moulds of some invasive fungal infections Galactomannan in aspergillus infection Culture Beta-glucan in aspergillus and candida Mainstay of diagnosis and identification Anti-microbial sensitivity testing technically Nucleic acid amplification - PCR more difficult than for bacteria May detect multiple types of fungi Increasingly used in specimens from Serology immunocompromised host Diagnosis of some fungal infections Very sensitive and may be difficult to distinguish colonisation from infection Copyright © Brunel University London v.3 2024. All rights reserved. Parasites Microscopy Most commonly used test for diagnosis examples include: Antigen detection Blood – malaria Rapid diagnosis of malaria on blood Urine- schistosomiasis samples – standard of care Stool – Amoebae and parasitic worms Available or in development for a range of other parasites but not in common use Culture Parasites cannot be cultured in vitro Nucleic acid amplification - PCR Not widely available Serology Increasing use in specialist centres Diagnosis of some parasitic infections Hard to interpret in endemic areas due to previous infections Copyright © Brunel University London v.3 2024. All rights reserved. Summary Many infections are diagnosed clinically and treated empirically Striving to achieve a definitive diagnosis by using tests to identify the organism has significant benefit to the patient and public health Microscopy, culture, serology, antigen detection and nucleic acid amplification are the tests most commonly used to identify organisms and their antimicrobial sensitivity patterns Copyright © Brunel University London v.3 2024. All rights reserved. Glossary Amoebae Candida Single celled free-living organisms. Can cause Yeast which is a common cause of minor gastrointestinal and liver infection infections but can also cause serious invasive disease in some settings Aspergillus Cerebrospinal fluid Fungal mould that causes a number of different Fluid that surrounds the brain and spinal cord illnesses in humans Cytomegalovirus Beta-glucan Virus from the herpes group. Infection is very Component of the fungal cell wall common and usually causes no or very mild symptoms. Serious invasive disease seen im Bone marrow the severely immunocompromised Found in the centre of long bones where blood Galactomannan cells are made. The bone marrow can be the site of a number of unusual infections Component of the fungal cell wall Herpes virus Bronchoscope Family of viruses that cause a number of Fibreoptic flexible device which is used to human illnesses visualise and take samples from within the bronchi in the lungs Copyright © Brunel University London v.3 2024. All rights reserved. Glossary Human immunodeficiency virus (HIV) Legionnaires disease Infects CD4 positive lymphocytes leading to Caused by a gram-negative bacteria, Legionella damage to the immune system. HIV was identified pneumophila. Causes pneumonia following as the cause of the acquire immunodeficiency inhalation of water containing legionella syndrome (AIDS) in the early 1980’s Malaria Immunoassay Illness cause by protozoal parasites (species of A technique that uses the highly specific binding of Plasmodia) transmitted by mosquitos. Mainly antibodies to antigens to identify substances found in tropical and sub tropical zones including microorganisms Meningitis Lumbar puncture Infection of the protective membranes, meninges, A technique which enable sampling of around the brain and spinal cord cerebrospinal fluid. A needle is passed between the vertebrae into the spinal canal below the level where the spinal cord ends. Copyright © Brunel University London v.3 2024. All rights reserved. Glossary Mycobacteria Streptococcus pneumoniae Group of bacteria including the organisms that Gram-positive diplococcus. Some strains are cause tuberculosis and leprosy highly virulent associated with sepsis, pneumonia and meningitis as well as infection at other body Neisseria meningitidis sites Gram-negative diplococcus that is one of the bacterial causes of meningitis. It can also cause Syphilis sepsis and infection at other body sites Sexually transmitted bacterial infection caused by a spirochaete – treponema pallidum Schistosomiasis Complex infection caused by parasitic worms – Tuberculosis species of schistosoma Pulmonary and systemic illness caused by Mycobacterium tuberculosis Smallpox Caused by the variola virus smallpox is a highly Window period transmissible and lethal infectious disease. The time between acquiring a pathogen and the Feared for millennia Smallpox is the first infectious presence of detectable antibody against that disease where vaccination has eradicated global organism transmission Copyright © Brunel University London v.3 2024. All rights reserved. To contact me: email: [email protected] Copyright © Brunel University London v.3 2024. All rights reserved.