11_diagnostics

• Manifestations of infection

  • host <-> microorganism interaction

  • virulence factors + host health

  • signs and symptoms dep. on body site and severity of infection

• microbial causes of infection

  • exogenous vs endogenous

• specimen selection, collection and processing

  • signs and symptoms as hints

  • collection of specific sample acc. to hypothetical specimen before any drug application

  • specimen amount + transport influencing lab results

• microbiologic examination

  • direct + indirect methods

Robert Koch Institute = government’s central biomedicine institute

-> identification, surveillance and prevention of (infectious) diseases via epidemiological and medical analyses

-> information provider and advisor of health-related political decision-making, scientific sector and general public

-> special laws for execution of tasks

-> federal health reporting (e. g. to WHO)

• consulting laboratories: guidance and advice for specific pathogen

• national reference centers: accurate diagnosis, resistance testing, prevention of spread, verification of tests from other labs, production and distribution of reference materials for external quality control and quality assurance

1. unlikely to cause human disease (E. coli K12)

2. human disease, probably hazard to employees, unlikely to spread, effective prophylaxis/treatment available (Burkholderia cepacia, Campylobacter jejuni, Corynebacterium diphtheriae, E. coli)

3. severe human disease, probably serious hazard to employees, may spread, usually effective prophylaxis/treatment available (Bacillus anthracis, E. coli O157, Mycobacterium tuberculosis)

4. severe human disease, serious hazard to employees, likely to spread, no effective prophylaxis/treatment available (Herpesvirus simiae, Ebola, Marburg virus, Congo haemorrhagic fever virus)

• broth

  • enrichment facilitates pathogenic growth of slow growing organisms in (low in volume) samples

  -> Mycobacterium tuberculosis, Haemophilus spp., Neisseria spp.

• differential media

  • specific dyes for quick identification due to characteristic colouring

• chromogenic agar

  • incooperation of multiple substrate-chromogen combinations w. careful interpretation due to eyeballing colouring (expensive)

• blood agar

  • rich, non-selective media for isolation of a range of fastidious bacteria

-> Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Clostridioides difficile, Proteus spp.

• growth inhibition of bacteria w. most effective drugs

MIC (Minimum Inhibitory Concentration)

• determination of lowest concentration of antimicrobial agent needed -> quantitative

• serial dilutions in grids + inoculum into microdilution plate in series after preparation of colonies -> no color: growth inhibited

Gradient diffusion method

• test strips w. antimicrobial agents into agar medium -> reading of concentration scale to determine susceptibility (Etest)

• correlated well w. MIC, but some systematic biases toward higher/lower MICs

Disk diffusion test

• similar to Gradiend diffusion method except using filter paper instead of test strips

• no accurate quantification of minimum antimicrobial concentration, no reliable results in combination w. some antibiotic/organism combinations

Automated MIC systems

• standardised w. suceptibility test results in short period of time

• detection of subtle changes in bacterial growth

• detection of bacterial antigens and antibodies

• only for specific bacterial infections

  • Legionella, Mycoplasma, Chlamydiae, Rickettsia, Borrelia

• no clearly identified epitopes possible

• specific IgMs only in acute phase of infection

indirect ELISA

• detection of antibody for pathogen

• find low-level infections

-> Borrelia burgdorferi, Treponema pallidum (Syphilis)

ELI-Spot (Enzyme-Linked Immuno Spot assay)

• looking for antibodies, measuring immune molecules and immune cells -> T-cell reaction against specific antigen w. colouring

-> chronic/recent Borrelia burgdorferi, Chlamydia pneuomonia, Chlamydia trachomatis, Yersinia species

PCR

• identification of pathogenic bacteria through 16S rRNA

• rapid diagnose infections via

  • ‘unique’ genes

  • high sensitivity and specificity

  • easy

  • rapid

mPCR (multiplex PCR)

• for small amounts of sample

• simultaneous amplification of multiple regions w. different primer pairs

  -> cost + time beneficial, exact diagnosis (sensitivity and specificity)

  -> reduced no. of separate reactions

• STIs primarily

  • Chlamydia trachomatis

  • Neisseria gonorrhoeae

  • Treponema pallidum

  • Trichomonas vaginalis

  • Mycoplasma genitalium

  • Ureaplasma urealyticum

  • Haemophilus ducrey

• limit: non-specific products - interference of primer-primer interactions w. amplification => decreased sensitivity + selectivity

LAMP-PCR (Loop-mediated isothermal amplification)

• recognision of distinct regions of target DNA -> subsequent rounds of amplification of those via loop structure

  -> increase of detection limits, efficiencyt, selectivity and specificity

• chorioaminonitis + premature labor

  • Ureaplasma parvum

  • Ureaplasma urealyticu

RT-PCR (Real-time PCR/qPCR)

• gold standard for detection and quantification

• avoiding cross contamination due to no further manipulation

• wide dynamic range for quantification

• determination of presence of specific genes and allels

  -> strand and isolates typing, AMR profiling, toxin production

• potential bioterrorism used pathogenic bacteria

  • Bacillus

  • Brucella

  • Burkholderia

  • Coxiella

  • Francisella

  • Yersinia

dPCR (digital PCR)

• direct quantification and clonally amplification of nucleic acids strains

• widely used for low-abundance nucleic acid detection + diagnosis of infectious diseases

• Preparation -> Partitioning -> Amplification -> Analysis

ddPCR (droplet digital PCR)

• method of dPCR

• good sensitivity for low-abundance DNA

• variety of infections (stage dependent)

• direct count of no. of bacteria

  • Borrelia burgdorferi

  • Staphylococcus

  • Salmonella

  • Listeria

  • Mycobacterium tuberculosis

• identification of bacterial pathogens at genus/species level

• spatial resolution, morphology information in mixed species samples

• biopsie or blood samples

• powerful

• direct incooperation of WGS in routine lab workflow

  • typing

  • presence of AMR genes

  • genes associated w. virulence and pathogenicity

• culture-free identification in complex polymicrobial samples

Metagenomics - Nanopore MinION

• portable, real-time DNA analysis for in-field, clinical deployment

• confident identification of

  • microbes to species level for accurate diagnosis

  • abundance within microbiota

  • AMR profiles

• NEC (preterm infant necrotising enterocolitis) - difficult diagnosis in early stage, associated w. sudden serious deterioration

  • Clostridium spp.

  • E. colit

  • Enterobacter spp.

  • Klebsiella spp.

  • MR/MAR

POCTs biosensors (Point of care tests)

• rapid detection + quantification of specific analytes by biorecognition elements (colorimetric/fluorimetric/electrochemical)

immune/AB based

• LFIAs (Lateral Flow Immuno Assays)

• several for bacterial infections

  • Myobacterium tuberculosis

  • multiplex for cystic fibrosis-associated ones - Pseudomonas aeruginosa, Staphylococcus aureus)

  • 10 endemic food-born pathogens - E. coli 0157:H7 , Parathyphi, Salmonella Typhi, Salmonella enteritidis, Vibrio cholerae O1/O139

  
  

nucleic acid-based

• often PCR based

• integration of multiple steps involved in bacterial detection onto a single chip

• multiple uses w. detection of specific bacteria and genes incl. toxins and AMR

• expensive, less/no flexibility

• Clostridioides difficle

protein and glycan-based

• reduction of unnecessary antibiotics treatment due to detection of microbial antigens

  • Neisseria gonorrhoeae

  • Staphylococcus aureus

• alternative use of host biomarkers for distinguishment btw. classes of infecting microbes

• MALDI-TOF-MS for routine identification of microbial pathogens

• advantage: direct identification from colonies grown on culture plates (less time consuming preparations needed)

• enhanced reliability and accuracy

• exploration of new perspectives

  • direct identification from positive blood cultures

  • sub-species typing

  • detection of drug resistance determinants

REIMS (Rapid evaporative ionization mass spectrometry

• quick and accuracte speciate bacterial due to species-specific liquid profile

• devlopment of customised platform for automation and high-throughput REIMS analysis

• iknife