Infectious Disease Molecular Diagnostics
Concepts
Understand nucleic acid amplification testing (NAAT) design for pathogens: target selection (conserved regions, multicopy genes for sensitivity), inclusivity (all relevant strains) and exclusivity (no cross-reactivity), and the role of internal controls. Know the difference between qualitative detection and quantitative viral load, and the clinical meaning of each. Be familiar with resistance genotyping (HIV, HCV, TB, CMV), and the interpretation of genotype-phenotype relationships.
Special considerations
Know the value of broad-range PCR (16S rRNA) and metagenomic sequencing, and their pitfalls (contamination, background, colonization vs infection). Understand the difference between a positive result in a sterile site vs a non-sterile site. Recognize the impact of low organism burden, inhibitors, and the need for a valid extraction control.
Traps
Items confuse detection with viability, misinterpret a positive result in a colonized site, or ignore the need for a quantitative standard. Watch for questions about resistance mutations that confer partial vs full resistance, and about the difference between a screening assay and a confirmatory assay.
Rule of thumb
For any infectious disease NAAT, require an internal amplification control to exclude inhibition, and interpret results in the clinical context (sterile vs non-sterile site, host immune status). A negative NAAT does not exclude infection if the specimen is inadequate or the pathogen burden is below the LoD.
Worked example
A respiratory sample is positive for a virus by multiplex PCR but the internal control shows late amplification. Repeat with dilution to relieve inhibition before reporting; a late internal control signals a potentially false-negative result for low-titer targets.