The tuberculin skin test (TST) became the gold standard for clinical diagnosis and identification of individuals with M. tuberculosis (MTB) infection (passive and active) after the publication of Robert Koch’s work (1891) with guinea pigs and the tubercle bacilli. The presence of a “wheal” reaction at the site of inoculation indicates a positive test requiring a repeat clinic visit for confirmation.
When testing individuals vaccinated with bacille Calmette-Guerin (BCG) vaccine or those who cannot return for reading of the TST, blood tests, such as interferon-gamma (IFN-g) release assays (IGRA)
, are available and recommended by the Food and Drug Administration (FDA).
- The IFN-g substance is present in the white blood cells of infected individuals and can be detected by IGRA tests, such as the QuantiFERON®-TB Gold Plus test and the T-SPOT® TB test.
- Disadvantages of the blood test method are the required laboratory processing within 8 to 32 hours of exposure (depending on method) and the failure of success in children less than five years or in immune-compromised persons.
- The test is preferred for anyone who received BCG or cancer therapy. BCG vaccine is usually given to children to prevent TB meningitis in developing countries where transmission of TB is high. (BCG is not recommended in the United States for pulmonary TB because of its questionable effectiveness and interference with the TST, which is used routinely.)
To date, many studies have reported evidence of an association between host genes and genetic polymorphisms, and TB susceptibility. Genes and mechanisms of resistance number many. The mechanisms, though not completely understood (and beyond the scope of this course), include decreased drug uptake, drug inactivation by constitutive beta-lactamases, increased efflux (e.g., fluoroquinolone resistance), alteration of the target site (inhibits RNA synthesis in RMP and mycolic acid biosynthesis, e.g. in INH), and reduced pro-drug-activating enzymes (pyrazinamide (PZA) resistance). This genetic component of resistance is useful in understanding and developing new genotyping procedures for testing resistance.
For epidemiological purposes, the genotyping of MTB strains has created an international database of information to connect patients, determine TB outbreaks, distinguish reinfection from reactivation, and identify laboratory contaminants. Programs for TB control in public health laboratories include genotyping, as directed by the CDC.
- The molecular technique, IS6110-restrictive fragment length polymorphism (RFLP), continues as the standard of reference for typing M. tuberculosis. The sequence, IS6110, present in numbers 0 to >20, is inserted at various loci on the genomes of MTBC isolates and used to compare patterns among laboratories.
- Spacer oligonucleotide typing (Spoligotyping), a PCR technique requiring small quantities of DNA, is another typing method.
- The newer, rapid typing MIRU-VNTR is based on variable numbers of tandem repeats (VNTR) of genetic element classes, or mycobacterial interspersed repetitive units (MIRU). MIRU-VNTR is more manageable but less discriminatory and may replace the current standard, the RFLP analysis.
- Whole-genome single nucleotide polymorphism (wgSNP) comparison, where wgSNP comparison is performed to identify single nucleotide polymorphisms (SNPs) that distinguish isolates. The SNPs can then be mapped on a genetic tree for comparison and to determine possible drug resistance.
- Whole-genome multilocus sequence typing (wgMLST), a genotyping scheme that uses whole-genome sequencing data to identify genotype clusters.
For rapid determination of drug-resistant Mycobacterium tuberculosis, molecular detection of drug resistance (MDDR) using conventional PCR and pyrosequencing (PSQ - a method of DNA sequencing)is currently offered by the CDC as part of public health laboratory TB control programs.