Clinical Virology & Diagnostics BMS2037 PDF

[email protected] www.maluquerlab.org @maluquerlab BMS2037: Clinical Virology & Diagnostics Dr Carlos Maluquer de Motes Reader in Molecular Virology Virology Lectures in BMS2037 • Introduction to Virology & Virus structure and Classification • Clinical Virology and diagnostics • Viro...

[email protected] www.maluquerlab.org @maluquerlab BMS2037: Clinical Virology & Diagnostics Dr Carlos Maluquer de Motes Reader in Molecular Virology Virology Lectures in BMS2037 • Introduction to Virology & Virus structure and Classification • Clinical Virology and diagnostics • Virology practical • The infectious cycle of virus replication mechanisms • Gastroenteritis viruses • Respiratory viruses • Zoonotic viruses Learning Outcomes • Understand why clinical virology is important • Why we monitor viral illnesses • Common techniques used in clinical virology • Pros and cons of different techniques • Understand the meaning of sensitivity, specificity, accuracy and precision. Why are virus diagnostics needed? 1. Appropriate management of patients • Avoids further unnecessary testing • Avoids unnecessary drug use (antibiotics!) • Informs patient treatment and prognosis • Are treatment strategies working (viral load testing) Why are virus diagnostics needed? 2. Routine public health measures • Screening of donated blood (HIV, HepB, HepC) • Notifiable infections (Measles, Rubella, Mpox, etc.) • Activate contact tracing • Limit spread or outbreak • Put mechanisms in place to contain and eradicate HEP B, C, E HIV Human T-lymphotropic virus (HTLV) Why are virus diagnostics needed? 3. Surveillance • To monitor the significance and prevalence of viruses in a community. • Monitoring and tracking of outbreaks (e.g. COVID-19) • Evidence of reemerging or emerging viruses – • Individuals travelling from other countries (e.g. polio in London). • Viruses spreading within animal communities (e.g. H5N1). • Viruses with zoonotic potential • Lecture on Zoonotic Viruses by Dr Petit. Viral infections share many symptoms • Diagnosis based on signs & symptoms is very difficult. • ‘Flu-like’ symptoms • • • • • • • Fever Chills Generalised aches and pains Headache Poor appetite Fatigue Drowsiness • ILI (Influenza-like illness) can be caused by: RSV, malaria, acute HIV, herpes, Hep C, rabies, dengue fever, pneumonia, measles, SARS, COVID-19 Surveillance-reported by diagnostic labs • UK Health Safety Agency (UKHSA) (former PHE). • Reporting weekly • Notifiable diseases • Non-notifiable diseases too. UKHSA COVID & flu report 13th Oct 2022 (other respiratory viruses) Sentinel surveillance of Influenza-like illness (ILI) • In addition to reported hospital testing • In Europe, ~5% of GPs are involved with sentinel surveillance • People attending the clinic with ILI have swabs sent to clinical virology labs • Monitors rates of circulating influenza and other respiratory viruses • Can see unusual patterns, monitoring of circulating sub-types FluSurvey (web-tool survey from self-reported respiratory symptoms from registered participants). FluDetector (internet-based search queries for ILI in the general population). Where did B/Yamagata go? Consequences? Clinical virology can help to identify new viruses • New viruses and new virus-disease associations are identified regularly! • SARS-CoV-2, COVID • Lujo virus • Over 90% of all human viruses known today were unknown at the end of World War II • Important for diagnosis and surveillance Clinical virology is dependent on good sampling! • The type of sample will largely be determined on the signs/symptoms of disease • This can indicate what organ systems are involved Methods of clinical virology • Isolation of virus • Cultivation in cell culture followed by identification Direct methods (detect infectious virus) • Detection of virus components • Of virions, viral antigens, viral nucleic acids • Serology • Detection of antibodies in the patient’s serum Indirect methods (cannot detect infectious virus) Isolation of virus • Viruses are OBLIGATE INTRACELLULAR PARASITES • What does this mean? Isolation of virus • Viruses need cells from other organisms to infect and replicate. • For new viruses very often trial and error to find out what the virus can infect-the ‘host range’ Cell lines (human, animal, insects) In Vivo (live animals) Embryonated eggs Cell culture- cytopathic effect H1N1 ATCC RSV Vanessa Ayala-Nunez, cytosmart Chin et al. 2016 Virus can be titrated (quantified) on living cells Plaque Assay Dilution series of virus on cells • Key technique in virology. • Time-consuming and not suitable for all viruses. • Mostly research technique, but still used in diagnostics (enrichment). • Metagenomic sequencing used when the target is unknown • If you know (or suspect the virus) you can use amplicon sequencing that amplifies a region of the viral genome • Sequences can be aligned to known virus sequences • If it is an unknown virus similarities and related viruses can be identified Direct Detection • Detection of virions, viral nucleic acids or viral antigens Definitions: Virion: A complete virus particle Viral nucleic acid: the genetic material of a virus, either RNA or DNA Antigen: molecular structures on the surface of viruses that can be recognized by the immune system Microscopy • Light microscopy- looking for consequences of infections, cytopathic effect. Staining tissue samples • Fluorescent microscopy-advances in staining techniques. Fluorescent tags binding to viral proteins • Electron microscopy- high resolution enough to visualize viruses Microscopy Schematic representation of immunohistochemical methods. A: direct method, B: indirect method, C: PAP complex procedure, D: ABC procedure, E: LAB procedure A HSVI immunoreactivity in the lung (IBD4 monoclonal antibody). B. LMP-1 immunohistochemistry showing positive staining in B cells (arrows) and plasma cells (arrow heads). Scale bar = 50μm. C. Immunohistochemical staining for HHV-8 LNA-1 in cutaneous patch/plaque Kaposi sarcoma. D. Immunohistochemistry of CMV showing nuclear positive cells in ileal tissue. x20 Example: Rabies • Rabies is caused by Rabies lyssavirus, transmitted in the saliva of infected animals. There is a wide host range and it has been detected in over 150 countries. • 55,000 die annually. • Early signs of rabies can be similar to other viral infections such as HSV1, VZV, West Nile virus & other viruses that cause encephalitis (inflammation of the brain). • Rabies has an incubation period of 2 weeks to 3 months and without treatment is (virtually) 100% fatal within a week of symptoms. • Treatment is post-exposure vaccination (5 vaccines and immunoglobulin)- very effective if given within days of exposure! Some signs of viral infection are obvious… Microscopy-Rabies WHO recommends fluorescent microscopy. PCR assay available. Diagnosis usually confirmed postmortem. The diagnosis can also be made from saliva, urine, and cerebrospinal fluid samples, but this is not as sensitive or reliable as brain samples. Cerebral inclusion bodies called Negri bodies are 100% diagnostic for rabies infection but are found in only about 80% of cases If possible, the animal from which the bite was received should also be examined for rabies (A) Direct immunofluorescence of fox brain, showing prominent green masses of viral antigen (Negri bodies). (B) Histopathology on human postmortem brain section. Finding Negri bodies (C) Immunohistochemistry used in research and in special diagnostic situations. Postmortem diagnosis in patients infected via organ transplant. viral antigen fills the axoplasm of peripheral nerves. Naphthol fast-red method. Scrubs: ‘My Lunch’ Electron Microscopy • Much better resolution than a light microscope. • A scanning transmission electron microscope has 50 pm resolution • Most light microscopes are limited to about 200 nm • Can view the structure of virions • During the 1970s new groups of hard to culture viruses discovered in faeces (rotaviruses, calciviruses, astroviruses, HepA) • One method: negative staining. Virus dilution on carbon coated grid. Virions adhere to the surface. Electron dense fluid added and surrounds virions. • Thin tissue sections can also be imaged. • Low sensitivity (need 106 virions/mL, ok for faeces but difficult for respiratory samples). • Impractical for large scale testing. https://journals.asm.org/doi/10.1128/CMR.00027-09 Influenza B pm= 1×10−12 m, or one trillionth (1/1000000000000) of a metre Nipah Viral nucleic acids: PCR • • • • • • Polymerase Chain Reaction Quick and (fairly) easy Can be easily scaled up for high-throughput testing Initially expensive, but cheaper in high volume Very sensitive and highly specific Can be quantitative (qPCR) • A positive PCR may not indicate active infection • Why do you think this might be? Advancements in PCR technology • PCR was first described in 1983 • A heat stable polymerase (Taq) was discovered in organisms that live in hot springs • Reducing environmental contamination • qPCR: quantitative • Multiplex PCR, in common use for diagnosis of viral infections Multiplex qPCR for viral detection • Different fluorophores can detect different viral nucleic acids. • The fluorescent signal for each fluorophore is measured • Commonly used for respiratory virus diagnosis Cycle Threshold (Ct) • The number of cycles of PCR needed until the sample is detectable. • The lower the number, the less cycles, the more viral nucleic acids present. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment _data/file/926410/Understanding_Cycle_Threshold__Ct__in_SARS-CoV-2_RT-PCR_.pdf COVID-19 PCR: Controversy or Conspiracy` “Big News-CDC withdraws PCR test from FDA EUA. It’s inability to differentiate Covid from Influenza was #1 reason.” The Truth: • In 2021 the CDC said: ”it encourages laboratories to consider adoption of a multiplexed method that can facilitate detection and differentiation of SARS-CoV-2 and influenza viruses.” • Cycle number too high? Serology • Serology is the scientific study of serum and other body fluids. In practice, the term usually refers to the diagnostic identification of antibodies or antigens in serum • Methods include: • • • • ELISA Agglutination Precipitation Complement fixation Detection of Viral Antigens: ELISA • Enzyme-linked immunosorbent assay (ELISA) or Enzyme Immunoassay (EIA) • Still used, but replaced by PCR in many instances. • An antibody to the target is absorbed on the solid surface. The unknown sample is added. If antigen (like viral protein) is present it will bind to the antibody. An enzyme labelled antibody is then added. A substrate is added. The enzyme, if present, will cause the substrate to change colour. • High sensitivity • Lots of different assay formats, easily adaptable Direct or Indirect ELISA Enzyme immunoassays (EIA or ELISA) for detection of virus and/or viral antigen. Left: Direct method. Right: Indirect method, using biotinylated antivirus antibody, followed by enzyme-labeled avidin. In each case an enzyme substrate is then added to develop a color reaction. Note the immobilization of the capture antibody on a solid support to facilitate subsequent washing steps Lateral flow tests • Immunochromatography available for HIV, dengue, influenza, COVID19, RSV (not all in common usage) • Antigens move through a support (filter paper, nitrocellulose film) • Labelled antibody reacts with sample containing antigen. • Second antibody produces a colour change • Can incorporate a positive and negative control • Less sensitive • Useful for rapid testing, point-of care testing Haemagglutination assay • Developed in the 1940s • Simple, relatively cheap, quick (a few hours) • Sialic acid receptors on RBCs bind to the HA protein on the surface of influenza and keep the RBCs in suspension. • No flu-RBCs settle Strengths & Limitations of clinical virology techniques Accurate & Precise • A good diagnostic test needs to be accurate and precise • Accurate: A test that provides a result close to the real value • Precise: A test is repeatable and the result reproducible • Cross-laboratory validation exercises Sensitivity and Specificity • Sensitivity: The probability of a positive test from a truly positive sample (true positive rate) • Specificity: The probability of a negative test from a truly negative sample (true negative rate) Key Points: • Appreciate the importance of clinical virology for surveillance, diagnosis, patient management and public health measures. • Understand some of the main techniques used in clinical virology within: • Virus isolation, direct detection and serology • Know pros and cons for different techniques • Learn key definitions (sensitivity, specificity, accuracy, precision, etc.) Some references (for your perusal): • Burrell, Howard, Murphy. 2017. Laboratory Diagnosis of Virus Diseases. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149825/ • Vajo & Torzsa. 2022. Extinction of the Influenza B Yamagata Line during the COVID pandemic – Implications for vaccine composition. Viruses 14:1745. • Paweska et al., 2009. Nosocomial outbreak of novel arenavirus infection, South Africa. Emerging Infectious Diseases 15:1598. Virology – useful sources Principles of Virology Fields Virology TWIV (podcast!) Viralzone http://viralzone.expasy.org/ All the Virology on the WWW http://www.virology.net/ The big picture book of viruses http://www.virology.net/Big_Virology/BVHomePage.html • International committee on taxonomy of viruses http://www.ictvonline.org/ • • • • • • Virology Lectures in BMS2037 • Introduction to Virology & Virus structure and Classification • Clinical Virology and diagnostics • Virology practical • The infectious cycle of virus replication mechanisms • Gastroenteritis viruses • Respiratory viruses • Zoonotic viruses