12) Diagnostics

Manifestations of Infection

• interaction between host and microorganism

• affected by host immune status and microbial virulence factors

• signs and symptoms vary according to site and severity of infection

  
  

Microbial Causes of Infection

• exogenous (acquired from environmental or animal sources or from other persons) or endogenous (from the ‘normal’ microbiota)

Specimen Selection, Collection, and Processing

• specimens selected on basis of signs and symptoms

• collected from medium representative of the disease process

• collected before administration of antimicrobial agents

• amount and rapidity of transport to lab influences test results

Microbiologic Examination

• direct and indirect methods (review previous lectures!)

Robert Koch Institute

• German government’s central biomedicine institution

• Identification, surveillance and prevention of diseases, especially infectious diseases

• analyses and evaluation of highly pathogenic and highly contagious diseases, which are of great significance to the general public

• Provides a scientific basis for political decision-making

• Executive tasks defined by special laws with regard to protection from infection

• Federal health reporting

• System of national reference centers (NRCs) and consultant laboratories (CLs)

Classification (Hazard group definitions)

• Class 1: unlikely to cause human diseasae

• Class 2: can cause disease, may be a hazard to emplyes but not to the community, prophylaxis/treatment available

• Class 3: can cause disease, serious hazard to emplyees, may spread to community, prophylaxis/treatment usually available

• Class 4: causes sever disease, serious hazard to employees, likely to spread to community, no prophylaxis/treatment

Differential:

• Mannitol salt agar (Staphylococcus): high salt concentration which inhibits growth of most bacteria

• MacConkey agar (Enterobacteriaceae): Lactose, bile salt

Chromogenic agar:

• Advanced differential agars

• Allow incorporation of multiple substrate-chromogen combinations

• Results have to be interpreted carefully

• Expensive

Blood agar

• Rich, non selective

• isolate different fastidious bacteria

AMR = antimicrobial resistance

• Susceptibility testing determines which antimicrobials will inhibit growth

• minimum inhibitory concentration (MICs):

  • assay: 2-fold serial dilutions of test compounds

  • Antimicrobial gradient method: gradient in agar medium from plastic strip

• Disk diffusion test

  • well standardised and has been widely evaluated

  • does not provide accurate information about MIC

Used to detect bacterial antigens and antibodies e.g. Legionella, Mycoplasma and Chlamydiae, Tick-born – Rickettsia and Borrelia

ELISA

• Antigen - specific Antibody - enzymy-conjugated secondary ab - substrate

• Absorbance correlates to concentraiton

Enzyme-Linked Immuno Spot assay (ELI-Spot)

• look for antibodies, measure immune molecules (e.g. cytokines) and immune cells

• Antibodys - Cell - cells produce cytokines - detection antibodies - avidin enzym conjugate

Serotyping

• used to identify bacterial strains (or serotypes)

• can be inaccurate, labor-intensive, and unreliable

Limitations

• Cross-reactions between the types of agents

• Some infectious agents have no clearly identified epitopes that are sufficiently specific

• Specific IgM antibodies are detected only in acute phase of infection, and for detecting active (not latent) infections

PCR

• can be used to target the 16S rRNA

• specific PCR assays based on unique genes to diagnose infections: high sensitivity and specificity, easy to perform, rapid

Multiplex PCR (mPCR)

• not limited by sample volume

• uses different pairs of primers

• minimises number of separate reactions -> allows accurate diagnosis of several diseases with a single diagnostic test

Loop-mediated isothermal amplification (LAMP-PCR)

• Alternative in clinical labs

• Used to detect species that cause chorioamnionitis and infections associated with premature labor

• uses primers recognising distinct regions of target DNA for a highly specific amplification reaction

• significant increase of detection limits, efficiency, selectivity, and specificity

Real-time PCR

• gold standard for detection and quantification of bacterial pathogens

• is safer avoiding cross contaminations (no manipulation of the samples is needed)

Digital PCR (dPCR)

• can directly quantify -> used for low-abundance detection

• Droplet digital PCR (ddPCR) -> used to diagnose bacterial infections that are sometimes hard to identify due to stage (low pathogen concentration)

Fluorescent in situ hybridisation – FISH

• Short fluorescence-labelled DNA bind to ribosomes of infectious agents → fluorescence microscopy

• Used for detecting bacteria in biopsies and blood cultures

• detect resistance-coding genes or resistanceassociated mutations in DNA

• extracted from purified bacterial isolates or directly from clinical samples

• faster than phenotypic methods

• limitations

  • can only detect known resistance genes or mutations

  • molecular and phenotypic test results are not always well correlated (may produce false negatives)

  • false-positives may be seen due to DNA contamination

• Powerful tool in medical microbiology

• Can be used for

  • typing of pathogens

  • presence of AMR

  • genes for virulence and pathogenicity

  • Culture-free identification of a range of pathogens in complex polymicrobial samples

Point of care tests (POCTs)

Biosensors

• based on the detection and quantification of specific analytes by biorecognition elements (colorimetric/fluorimetric or electrochemical)

• implemented in community, primary care and hospital settings

Immune (antibody)-based

• Focus on Lateral Flow Immuno Assays (LFIAs

nucleic acid-based

• often PCR based

protein and glycan-based

• Detection of microbial antigens as indicators of infection

• or of host biomarkers to distinguish classes of infecting microbes (e.g. bacterial vs viral)

MALDI-TOF MS

• identifying bacteria directly from colonies in a few minutes

• relieable and accurate

• possibly identification of pathogens directly from positive blood cultures, sub-species typing or detection of drug resistance determinants

Rapid evaporative ionization mass spectrometry (REIMS)

• identification based on speciesspecific lipid profile