Genotyping, Resistance, and Molecular Epidemiology

Genotype to choose treatment

Detecting an organism tells you what’s there. Genotyping tells you which strain and whether it carries resistance, so the drugs can be chosen before (or instead of) slow culture-based susceptibility testing.

HIV drug resistance

HIV’s reverse transcriptase makes errors constantly, and under drug pressure resistant variants take over. Genotypic resistance testing amplifies and sequences the HIV genes for drug targets - reverse transcriptase, protease, and integrase - and reports mutations linked to resistance (for example, M184V in reverse transcriptase reduces susceptibility to lamivudine and emtricitabine, and K103N causes high-level resistance to efavirenz and nevirapine).

When and how ¹:

• At entry into care, to detect transmitted resistance.
• At virologic failure, while the patient is still taking the failing regimen or within 4 weeks of stopping it. Off drugs, the wild-type virus regrows and resistant variants fall below detection.
• The viral load must be high enough: recommended above 1,000 copies/mL, and worth attempting at 500 to 1,000 ¹.

Standard Sanger-based genotyping detects variants making up roughly 20% or more of the viral population; minor variants can be missed.

Phenotypic testing (measuring viral growth in drug) exists but is slower and costlier, used mainly for complex resistance.

Hepatitis C genotype

HCV has several genotypes (1 through 6 are common, with subtypes such as 1a and 1b). Genotype once dictated drug choice and duration. Modern pangenotypic regimens work across genotypes, so genotyping is now needed mostly for patients with prior treatment failure, cirrhosis, or regimens that require it. HCV resistance-associated substitution testing is used in selected cases.

Bacterial resistance genes

Molecular detection of resistance genes gives answers in hours:

• mecA / mecC: methicillin resistance in staphylococci ².
• vanA / vanB: vancomycin resistance in enterococci (VRE).
• Carbapenemases: the five most common mobile carbapenemase genes in Enterobacterales are KPC, NDM, VIM, IMP, and OXA-48 ³. Carbapenemase-producing organisms resist nearly all beta-lactams and spread between species on plasmids, so detecting them triggers infection control action.

The limit: a gene panel only finds the genes it looks for. Absence of a gene doesn’t prove susceptibility, because resistance can come from other genes, mutations, efflux pumps, or porin loss. Phenotypic susceptibility testing still matters.

Mycobacteria

Rapid molecular tests detect Mycobacterium tuberculosis complex directly from sputum and simultaneously detect rifampin resistance from mutations in the rpoB gene. Because rifampin resistance usually accompanies isoniazid resistance, it’s a marker for multidrug-resistant TB.

Molecular epidemiology

Genotyping also answers: are these cases connected?

• Pulsed-field gel electrophoresis (PFGE) cut bacterial genomes with a rare-cutting restriction enzyme and compared band patterns. It was the standard for decades.
• Whole genome sequencing (WGS) compares entire genomes, down to single-nucleotide differences. In 2019, CDC’s PulseNet, the national foodborne outbreak network of public health labs, completed its transition from PFGE to WGS ⁴. In a 2018 Salmonella outbreak linked to ground beef, WGS showed that some cases sharing the outbreak PFGE pattern weren’t actually part of it ⁴.

Hospitals use the same approach to investigate clusters of MRSA, VRE, CRE, and C. difficile: closely related genomes suggest transmission; distant ones point to separate introductions.

What to take from this

HIV genotyping sequences reverse transcriptase, protease, and integrase, needs about 1,000 copies/mL, and should be done on the failing regimen or within 4 weeks of stopping. HCV genotyping matters less with pangenotypic drugs. Resistance gene tests (mecA, vanA/B, KPC, NDM, VIM, IMP, OXA-48, rpoB) give fast answers, but a missing gene doesn’t prove susceptibility. Whole genome sequencing has replaced PFGE for outbreak investigation.