Lecture 2: Diagnosis of Infectious Disease PDF

Lecture 2 Diagnosis of infectious disease Dr. Claire Atkinson Why do we need to diagnose an infection? Why do we need to diagnose an infection? Determine the infectious agent Management of infection o Appropriate treatment o Confirm the treatment is working – is it not detected after treatment Infec...

Lecture 2 Diagnosis of infectious disease Dr. Claire Atkinson Why do we need to diagnose an infection? Why do we need to diagnose an infection? Determine the infectious agent Management of infection o Appropriate treatment o Confirm the treatment is working – is it not detected after treatment Infection control o Prevent the spread of infection o Inform on infectivity Diagnostic Questions ? Am I infected? What am I infected with? Can I be treated? Is so what with and for how long? Is my infection improving or showing signs of becoming more virulent? Am I infectious to others? Have I been infected in the past? Am I immune to infection? Diagnostic Questions ? Am I infected? What am I infected with? Can I be treated? Is so what with and for how long? Is my infection improving or showing signs of becoming more virulent? Am I infectious to others? Have I been infected in the past? Am I immune to infection? Laboratory tests can answer all these questions Samples: Blood, saliva, urine, pus, vomit, feces, tissue biopsies, mucosal swab ……………… Specimens should reflect the disease process and be collected in sufficient quantity to allow complete microbiologic examination. ‘Put the wrong information in and get the wrong results out’ Skin and mucous membranes have a large and diverse indigenous flora, every effort must be made to minimize specimen contamination during collection. Specimens collected by invasive techniques, particularly those obtained intraoperatively, require special attention. Enough tissue must be obtained for both histopathologic and microbiologic examination. If possible, specimens should be collected before the administration of antibiotics. Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David A. Stahl 2022 How are infections diagnosed in the lab? Key questions….. How fast do we need the result? Can we propagate the pathogen easily? How dangerous is the pathogen? Might it be easier to detect an immune response? Many bacterial and fungal infections are diagnosed by propagating them on agar plates Direct Diagnosis: Microscopy: Staining, Immunofluorescence and other immunoassays may detect specific microbial antigens. Culture: Isolation of infectious agents frequently requires specialised media. Selective media contain inhibitory substances that permit the isolation of specific types of microorganisms. MALDI-TOF of isolates Microbial Identification: Colony and cellular morphology may permit preliminary identification. Growth characteristics under various conditions, utilization of carbohydrates and other substrates, enzymatic activity, immunoassays, and molecular methods such as PCR are also used. Antimicrobial Susceptibility: determine susceptibility to antimicrobial agents. Indirect Diagnosis: Serodiagnosis: A high or rising titer of specific antibodies may suggest or confirm a diagnosis. Direct Diagnosis: Microscopy: Staining, Immunofluorescence and other immunoassays may detect specific microbial antigens. Differences in cell wall structure/ bacterial morphology Gram positive = Purple Gram Negative= Pink Direct Diagnosis: Microscopy: Staining, Immunofluorescence and other immunoassays may detect specific microbial antigens. Direct Diagnosis: Microscopy: Staining, Immunofluorescence and other immunoassays may detect specific microbial antigens. Immunofluorescence of nasopharyngeal secretions. FITC stain = Green Colour Electron microscopy of nasopharyngeal swab Diagnosis? Adenovirus respiratory infection Direct Diagnosis: Culture: Isolation of infectious agents frequently requires specialised media. Selective media contain inhibitory substances that permit the isolation of specific types of microorganisms. MALDI-TOF of isolates for confirmation (if required). Differential and or selective agars – depending on sample type Mannitol Salt agar Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David A. Stahl 2022 McConkey Agar Direct Diagnosis: Culture: Isolation of infectious agents frequently requires specialised media. Chromogenic agar contain substances that permit the isolation of specific types of microorganisms based on colour changes. MALDI-TOF of isolates for confirmation (if required). Chromogenic agars – depending on sample type Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David A. Stahl 2022 Matrix-assisted laser desorption/ionization time of flight (MALDI- TOF) (1) Sample protein molecules are converted into ions in the gas phase and measured for their mass-to-charge (m/z) ratio. (2) A laser strikes the sample converting the protein molecules into gas (3)The ionized molecules are subsequently accelerated by a potential difference and fly through the high-vacuum flight tube towards the detector. (4)The system measures the analytes ‘time of flight’ to the detector and produces a characteristic spectrum. Lighter ions take less time to travel to the detector; heavier ions take more time. (5) The software identifies the sample by comparing the resulting pattern of mass peaks to a library of known patterns. Matrix-assisted laser desorption/ionization time of flight (MALDI- TOF) In simple terms Ø A smear of a sample is inoculated onto a steel plate Ø A small amount of matrix solution is added and allowed to dry Ø The target is then introduced into the fully automated MS system Ø Species ID is determined from the protein peaks of the generated spectrum Direct Diagnosis: Microbial Identification: Colony and cellular morphology may permit preliminary identification. Growth characteristics under various conditions, utilization of carbohydrates and other substrates, enzymatic activity, immunoassays, and genetic probes are also used. The ability of microorganisms to utilize certain biomolecules, resulting in useful organic compounds for themselves forms the basis of various biochemical tests Antimicrobial Susceptibility: determine susceptibility to antimicrobial agents. Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David A. Stahl 2022 Urine sample - brining it all together So what happens if you can’t grow the pathogen? Many bacteria take too long to grow or have complex growth requirements Viruses cannot be grown, require complex cell culture systems with specific cell types Protozoa and helminths often have complicated life cycles with more than one host so cannot be grown in culture How do you diagnose? How do you predict the pathogens susceptibility? What sort of test could we use? So what happens if you can’t grow the pathogen? How do you diagnose? How do you predict the pathogens susceptibility? What sort of test could we use? By detecting a component of the pathogen by immunological, biochemical or genomic means However, caution must be taken as this does not mean that the pathogen is infective or even the causative agent Point of care tests Antigen detection – agglutination test Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David A. Stahl 2022 Genomic molecular methods Detection of nucleic acid DNA/RNA Uses sequence complementarity to detect specific targets If sequences match then they will bind to each other –hydrogen bonds Molecular methods: Polymerase Chain Reaction 3 stages Can multiple billions of copies of DNA in 30 cycles visualise of gel or in realtime Molecular methods: Polymerase Chain Reaction What if you want to detect an RNA virus or ribosomal RNA? RNA needs to be converted into cDNA via a process called reverse transcription à Followed by PCR Retroviruses contain RTase and have a RT Step as part of their life cycle What if you want to know how much pathogen is present? Quantitative real-time PCR Sensitive, specific and no need for isolation What if you want to know how much pathogen is present? Quantitative real-time PCR Pathogen load can be determined by running against known concentration of target à generate standard curve à interpolate unknown What if you want to know how much pathogen is present? Applications of qPCR - viral loads (HIV, HBV, HCV, CMV) Pre-emptive therapy post transplant - CMV HIV Viral Load But PCR is an extremely powerful technique! It is extremely sensitive and can be easily contaminated It requires specialist equipment Can be expensive Needs expertise to set-up and run ‘It can still give a positive result even when the pathogen is no longer replicating and the patient is no longer infectious’ Molecular methods: sequencing Resulting chromatogram shows DNA sequence, which can be input into databases of known sequences to give antiviral resistance/ ID of unknown organism/ mutational analysis. Genomics: 16s Ribotyping (rRNA sequence dependent) (A) Bacterium contains circular DNA and a ribosome composed of 2 subunits, the smaller of which contains 16S rRNA. (B) Within bacterial DNA, the 16S rRNA gene, which codes for the 16S rRNA utilized in ribosomes, has been extremely well conserved throughout evolution because of its necessity for bacterial protein synthesis. (C) Because it has been so well conserved, the 16S rRNA gene is specific for bacterial species. It can therefore be amplified with PCR and sequenced to determine the types of bacteria present (D) Organismal classification system and (E) example of classification system. Created with BioRender.com Genomics can characterize pathogens and help control the disease they cause Genomics can characterize pathogens and help control the disease they cause Application of WGA: SARSCov-2 surveillance VOI – variant of interest VOC – Variant of concern Indirect Diagnosis: Cultivating a pathogen directly is fine if you know what specimen to take…will grow and is sufficient in quantity What do you do if you don’t know? Or you want to prove immunity due to past infection/ vaccination Serodiagnosis: A high or rising titer of specific antibodies may suggest or confirm a diagnosis, prove past infection or protection from vaccination Known as serology Diagnostic methods - serology Following exposure antibodies will start to appear in most patients 1st IgM 2nd IgG It is the indirect detection of these antibodies that is used routinely to diagnose viral infection Immune assays timing Immune assays –ELISA enzyme linked immunosorbent Assay Laboratory automation is now commonplace High throughput of samples Can hep reduce analytical error Syndromic testing ‘Syndromic diagnostic testing is a novel approach to the rapid diagnosis of common infectious diseases, including bloodstream, respiratory, gastrointestinal, and CNS infections As the global burden of antimicrobial resistance continues to rise, the judicious use of antimicrobials is of utmost importance’ Syndromic testing - example The BioFire® FilmArray® Gastrointestinal (GI) Panel https://www.biomerieux.com/corp/en/our-offer/clinical-products/biofire-respiratory-2-1panels.html Syndromic testing - example The BioFire® FilmArray® Gastrointestinal (GI) Panel Syndromic testing - example https://www.biofiredx.com/blog/value-syndromic-testinggastroenteritis/ BUT…… £££££ Multiplex PCR Many labs run multiplex PCR to reduce time to diagnosis ‘Multiplex PCR refers to the use of polymerase chain reaction to amplify several DNA sequences simultaneously’ Can be used to detect multiple targets or with an internal Control to measure inhibition of the PCR reaction. SARS CoV19 removed the need for RNA extraction of swabs https://www.biorxiv.org/content/10.1101/2020.04.06.028316v2.full.pdf+html Questions BREAK Tutorial Select the best diagnostic test- case studies Measles outbreak UK 2023 A 26 year old teacher presents to the GP after being in contact with a case of measles at his school, he is clinically well What test would be the best to run? What would you ask the GP? What information can you give the patient from the results? Test results: Measles IgM negative Measles IgG positive What information can you give the patient from the results? A sore throat A 15 year old, presents to the GP with a sore throat. On clinical examination they have a fever, pain when swallowing and are diagnosed with pharyngitis. What sample would you take? What test would be the best to run? Sample required – charcoal swab Grow on blood agar – shows beta haemolysis (Complete lysis of red celles around the colony) Lancefield group A = Streptococcus pyrogenes is the most common cause of a sore throat Antibiotic choice = penicillin or amoxicillin