MOLECULAR DIAGNOSTICS
S. pneumoniae is the polysaccharide capsule as it interferes with host opsonophagocytic clearance mechanisms, making it the primary target of many pneumococcal vaccines. As there are over 100 known polysaccharide serotypes and protection is strictly serotype-specific, developing effective vaccination strategies can be challenging. Pneumococcal conjugate vaccines (PCVs) which immunise against specific serotypes are typically used to protect against S. pneumoniae infections, and ongoing research looks to expand formulations to offer broader serotype coverage. For example, the PCV10 vaccine, approved by the European Medicines Agency (EMA) in March 2009, targets the 10 most common disease-causing serotypes, while the latest PCV vaccine (PCV21) covers 21 different strains, with a PCV24 vaccine currently in development. Continuous surveillance of strain and
serotype diversity is therefore essential to ensure that vaccine formulations target the most prevalent disease-causing serotypes, conferring protection to those immunised and limiting the transmission of threatening invasive disease.
Traditional methods Culture-based workflows, biochemical testing and antimicrobial susceptibility testing (AST) are well established methods for the identification of bacteria at species-level and can be used to perform basic phenotyping. Although they are highly reliable, these methods are also time and labour intensive. It can take up to 72 hours to obtain results using culture-based workflows, which can significantly delay the treatment of severe infections that demand fast response times. Furthermore, workflows can be lengthy and require manual intervention at several key stages, which is a particular challenge for reference laboratories handling large workloads. Lacking the ability to perform
serotyping and strain-level characterisation, these methods are often used alongside molecular and
Culture-based workflows are highly reliable; but also time and labour intensive, taking up to 72 hours to obtain results.
immunological approaches. Molecular techniques, such as polymerase chain reaction (PCR)-based serotyping and whole-genome sequencing (WGS), offer rapid and sensitive microbial analysis but are costly due to expensive reagents and equipment. They also rely on large infrastructure, including specialised hardware and bioinformatics systems, which must have the capacity to manage high-throughput workflows. The historical gold standard for
S. pneumoniae serotyping is the Quellung reaction (Neufeld test): a method that uses the immunological properties of capsular epitopes to identify pneumococcal capsular types. The technique allows for visual confirmation of the presence of bacterial capsules and provides highly specific results. However, it is costly, time- consuming and dependent upon skilled staff with extensive experience and
training. A rapid, standardised and more cost-effective method that can differentiate below the species level is needed to support routine public health S. pneumoniae surveillance.
Complementary phenotypic platforms Mass spectrometry (MS) profiling used alongside FTIR spectroscopy provides comprehensive microbial identification at both species and strain level. Matrix-assisted laser desorption/ ionisation-time of flight (MALDI-TOF) MS generates unique proteomic spectral fingerprints of unknown microorganisms which are then matched against a large reference library to determine the identity of the species. Samples are processed within minutes, enabling rapid identification from a pure culture. Offering reproducible and cost-effective analysis, MALDI-TOF MS is well established in routine microbiology workflows. Following species confirmation with
MALDI-TOF mass spectrometry generates unique proteomic spectral fingerprints of unknown microorganisms which are then
matched against a large reference library to determine the identity of the species
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MALDI-TOF MS, FTIR spectroscopy can be used to differentiate below the species level, resolving differences among strains, isolates or clones. The technique analyses the molecular vibrations caused by the absorption of infrared light across various cellular components, including carbohydrates, lipids and proteins. As different chemical structures vibrate at different wavenumber regions, it provides information about the full range of diagnostic molecules present in the sample. The carbohydrate
By Gsbhalla - Own work, CC BY-SA 4.0
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