12_Diagnostics

• Susceptibility testing is used to determine which antimicrobials will inhibit the growth of bacteria causing a specific infection

• Results from this test will help a healthcare practitioner determine which drugs are likely to be most effective in treating an infection

Used to determine the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism

• Employs thin plastic test strips that are impregnated on underside with a dried antibiotic concentration gradient and are marked on upper surface with a concentration scale

• MIC is determined by the intersection of the lower part of the ellipse shaped growth inhibition area with test strip • Etest results correlate well with MICs

• however, some systematic biases toward higher or lower MICs

minimum inhibitory concentration

Based on observation that strains of the same species can differ in their antigenic determinants

• Classic tool for identifying bacterial strains (or serotypes)

• Applied to numerous bacteria that express different serotypes

• Salmonella

• Pseudomonas aeruginosa

• E. coli

• Shigella

• Yersinia

• Vibrio cholera

In diagnostic laboratories the use of PCR is often limited by cost and availability of adequate sample volume

• To overcome these issues mPCR uses different pairs of primers to simultaneously amplify multiple regions of the nucleic acid of the sample

• significant benefits in cost, time, and exact diagnosis

• Minimises number of separate reactions

• detect several pathogens at the same time in single specimen e.g. sexually transmitted pathogens

• Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, Ureaplasma urealyticum, Haemophilus ducrey

• Technique allows diagnosis of several diseases with a single diagnostic test, with sensitivity, specificity, and speed

• indispensable values in diagnostic tests!

• Limitations such as nonspecific products generated through primer-primer interactions may interfere with amplification of targets

• decreasing sensitivity and selectivity of reactions

Alternative to PCR-based methodologies in clinical labs

• uses 4-6 primers recognizing 6-8 distinct regions of target DNA for a highly specific amplification reaction

• strand-displacing DNA polymerase initiates synthesis and 2 of the primers form loop structures to facilitate subsequent rounds of amplification

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

• Used to detect species that cause chorioamnionitis and infections associated with premature labor, Ureaplasma parvum and Ureaplasma urealyticum

Real-time PCR

Molecular AMR diagnostics detect resistance-coding genes or resistance-associated mutations in DNA extracted from purified bacterial isolates or directly from clinical samples

Much faster results than phenotypic methods

Global Antimicrobial Resistance Surveillance System (GLASS)

Need for rapid diagnosis has resulted in the development of novel diagnostic devices based upon the detection and quantification of specific analytes by biorecognition elements

• colorimetric/fluorimetric or electrochemical

• implemented in community, primary care and hospital settings

Different forms:

• POCTs – immune (antibody)-based

• POCTs – nucleic acid-based

• POCTs – protein and glycan-based (Detection of microbial antigens as indicators of infection by specific pathogens, Alternative is the use of host biomarkers to distinguish classes of infecting microbes)