Diagnostics of Mycobacterium

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.
Antimycobacterial agent Genetic marker
Primary agents
Rifampin rpoB (RNA polymerase subunit)
Isoniazid katG (catalase-peroxidase)
inhA (enoyl-acyl carrier protein reductase)
ahpC (alkyl-hydroperoxide reductase)
kasA (keto-acyl synthase)
Ndh (NADH dehydrogenase)
Pyrazinamide pncA (pyrazinamidase)
Streptomycin rpsL (S12), rrs (16S rDNA)
Ethambutol embB (arabinosyl transferase)
Secondary agents
Capreomycin
Kanamycin
Cycloserine
Ethionamide inhA (enoyl-acyl carrier protein reductase)
Alternative agents
Rifapentine rpoB
Rifabutin rpoB
Amikacin
Quinolones, ciproflaxin gyrA, gyrB (DNA gyrase)
Genetic targets relevant for direct detection of
antimycobacterial drug resistance development