Diagnostics of Mycobacterium Initial screening: TB skin test (Purified Protein Derivative).

1. Diagnostics of Mycobacterium • Initial screening: • TB skin test (Purified Protein Derivative). • Drawbacks: BCG injected subjects are positive, 3 days delay for result • QFT-G test (measures INF- response to TB specific antigen) • Active TB tests, depending on the suspected location of bacterium: • 3-5 samples of sputum • multiple gastric aspirate • urine (UTI) • CSF (meningeal) • Cultures • Samples are processed for fast acid stain (FAS smear positive indicates Mycobacterium) and cultured after alkali decontamination (30s in 1-2% NaOH) • Molecular methods use species-specific genes, including light and heave ribosomal RNA Clinical specimen/ decontamination culture • Direct detection: • Microscopy • PCR • MTB rifampin resistance • Species identification: • 16S rRNA hybridization (MTB and MAC) • 16S rRNA gene PCR sequencing (NTM) • restriction fragment length polymorphism Susceptibility testing Rifampin resistance (PCR oligohybridization sequencing)

2. Drug sensitivity The raise of drug-resistance strains makes strain identification necessary for the selection of the proper pharmacological treatment. Several genes have been found to confer specific drug-resistances. Such genes are now readily identified using molecular techniques (most of them PCR-amplification based), at least for the already sequenced drug-resistance associated genes. Identification and characterization of unknown types of resistance requires de-novo studies. Genetic targets relevant for direct detection of antimycobacterial drug resistance development