November30th,2019Michelle Dotzert, PhD
Diagnosis of microbial infections requires specimen collection, sample preparation, and analysis. New methods are emerging that allow for faster, more cost-effective, and more accurate microbial identification. Quicker diagnosis allows for fast treatment and prevents the overuse of antibiotics, which can lead to resistance.
Standard procedure for confirming an infectious disease requires specimen collection and test selection. Routine diagnostic tests include microscopic examination, culture and biochemical tests, serological tests including agglutination and ELISA, and genetic tests. More advanced diagnostic methods are emerging, focused on speed, accuracy, and cost effectiveness.
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)
MALDI-TOF-MS has been approved by the FDA for microbial identification. The Clinical and Laboratory Standards Institute Guideline M58 published in 2017 provides information for sample preparation and analysis, result interpretation and reporting, and troubleshooting.
MALDI-TOF-MS is a nonfragmenting, or soft ionization, technique. The analyte is embedded in an acidic matrix material on a metal plate, and nitrogen laser excitation is used to catalyze the charge transfer from the matrix to the analyte for desorption. Ions are separated based on their m/z, and a mass analyzer is used for detection and creation of a spectral profile. Future directions for MALDI-TOF-MS include antimicrobial susceptibility testing, microbial virulence, and glycans.
Next-Generation Sequencing (NGS)
The NGS workflow begins with pathogen culturing and isolation, followed by DNA extraction and library preparation. Images or signals are converted into base calls during primary analysis. Further data processing in secondary analysis includes trimming and filtering, and sequence reads are assigned to a reference sequence or assembled with de novo assembly. The identification of clinically significant findings in tertiary analysis is used to generate a final report.
Automated Polymerase Chain Reaction (PCR)
Multiplex PCR is especially useful for specimens from patients presenting with nonspecific symptoms, which may result from any number of different pathogens. A sample-to-result automated PCR system enables the addition of a clinical specimen directly to the device. The sample is treated with multiple reagents for nucleic acid extraction followed by amplification and detection of a target sequence. Platforms range in classification from high-complexity molecular assays to FDA-cleared moderate-complexity IVD tests.