Molecular Diagnosis Techniques

Amplifies DNA target using primers, dNTPs, Taq polymerase
Steps: Denaturation → Annealing → Extension
Detection via gel or fluorescence in qPCR

How does PCR work in detecting CNS infections?

Uses fluorescent-tagged dNTPs
Quantifies DNA based on Ct (Cycle threshold) value

What is the principle behind real-time (qPCR)?

Isothermal: No thermocycler needed
Uses loop-forming primers for amplification
Faster and simpler, used in point-of-care settings

How does LAMP differ from PCR?

Sequence-based method to detect all DNA (host + pathogen) in CSF
Useful for undiagnosed infections or rare pathogens (e.g., tapeworm in 2017 case)
Requires bioinformatics to interpret microbial community

What is metagenomic sequencing and its benefit in CNS infections?

Detects unexpected or novel pathogens

What are the advantages of metagenomics?

Expensive, requires high bioinformatics expertise
May be less sensitive without sufficient DNA

What are the limitations of metagenomics?

Based on peptide mass fingerprinting
Identifies organisms based on their protein profiles
Does not require culture if CSF is sterile

What is the principle of MALDI-TOF in pathogen identification?

Rapid identification
Can detect some antimicrobial resistance-associated proteins

What is a key benefit of MALDI-TOF over traditional culture?

Identifies individual immune cells and their gene expression
Locates cells spatially in CNS (important in diseases like HIV encephalopathy)
Detects immune exhaustion (e.g., CD4+ T-cell dysfunction)

What does single-cell multi-platform analysis offer in CNS research?

Uses fluorescent-labeled antibodies against CD markers
Can detect up to 64 different markers in a single run

How does flow cytometry identify immune cell types?

DNA-protein lattices released by neutrophils to trap microbes
May contribute to tissue damage if unregulated
Transcriptomic analysis shows distinct protein profiles for harmful NETs

What are NETs and their role in CNS pathology?

Most current reference genomes are based on Caucasian populations
Genetic variations in underrepresented groups (e.g., Asians) may be missed
Limits the effectiveness and equity of diagnostics in LMICs like the Philippines

Why is diversity important in reference genomes for infectious disease diagnostics?