What is tuberculosis (TB) genotyping?
TB genotyping is a laboratory-based approach used to analyze the
genetic material (e.g., DNA) of Mycobacterium tuberculosis, the
bacteria that cause TB disease. The total genetic content is
referred to as the genome. Specific sections of the M. tuberculosis
genome form distinct genetic patterns that help distinguish
different strains of M. tuberculosis.
Why use TB genotyping?
TB genotyping results, when combined with epidemiologic data,
help identify persons with TB disease involved in the same chain of
recent transmission. In the same way, TB genotyping helps
distinguish between persons whose TB disease is the result of TB
infection that was acquired in the past, as compared to recently or
newly acquired infection with development of TB disease. TB
genotyping is a tool that can add value to conventional contact
investigation. Below are some applications:
- Discover unsuspected transmission relationships between TB
- Identify unknown or unusual transmission settings, such as bars
or clubs, instead of traditional settings like home and workplace
- Uncover inter-jurisdictional transmission
- Establish criteria for outbreak-related case definitions
- Identify additional persons with TB disease involved in an
- Determine completeness of contact investigations
- Detect laboratory cross-contamination event
- Distinguish recent infection (with development of disease) from
activation of an old infection
Since TB prevention and control efforts directed at preventing TB
transmission are fundamentally different from efforts to prevent
activation of latent TB infection, genotyping offers a powerful tool
to help direct the application of appropriate efforts. Furthermore,
TB genotyping allows us to monitor our progress toward eliminating
TB transmission more accurately.
What is the difference between a specimen and an isolate?
A specimen is a clinical sample (e.g., sputum, bronchial wash,
urine, blood, or cerebrospinal fluid; tissues from organs or gastric
aspirates) from a patient suspected of having TB. Specimens may or
may not contain M. tuberculosis. If M. tuberculosis
grows in culture
media, it is called an isolate. Only isolates identified as M.
tuberculosis can be genotyped.
The methods used by the Centers for Disease Control and
Prevention (CDC) contract genotyping laboratories will not genotype
Mycobacterium avium or other non-tuberculous mycobacteria.
What laboratory techniques and methods are used in the National
TB Genotyping Service (NTGS)?
CDC contracts with two genotyping laboratories to provide the
NTGS. These laboratories routinely use two polymerase chain reaction
(PCR) methods on all M. tuberculosis
oligonucleotide typing (spoligotyping) and variable-number tandem
repeat of mycobacterial interspersed repetitive units (VNTR-MIRU).
These methods yield digital results that can be readily analyzed.
Click here for a
detailed description of these methods.
- Spoligotyping: Identifies the M. tuberculosis
genotype based on
presence or absence of spacer sequences found in a direct-repeat
region of the M. tuberculosis
genome where 43 identical sequences
and 36 base pairs are interspersed by spacer sequences. This highly
reproducible method gives results in a standardized 15-digit code
that can be easily analyzed and communicated between laboratories
and TB programs (e.g., 777777777760771).
- VNTR-MIRU: Distinguishes the M. tuberculosis
strains by the
difference in the number of copies of tandem repeats at specific
regions, or loci, of the M. tuberculosis
genome. Like spoligotyping,
this typing method yields results in a standardized code that can be
easily analyzed and communicated between laboratories and TB
programs. A total of 41 MIRU loci have been reported; however, most
laboratories target only 12 loci. Newer versions of the method at
the NTGS laboratories now include 24 loci, which may increase
discriminatory power. When laboratories employ the 12-loci method,
the results are a 12-digit code (e.g., 223225163324); 24-loci method
results in a 24-digit code (e.g., 223225163324561333245623).
A third method, IS6110-based Restriction Fragment Length
Polymorphism (IS6110-RFLP), may be utilized upon special request. Click here for
details on when to request RFLP.
- IS6110-based RFLP: This method detects variations in a specific
section of the M. tuberculosis
genome called insertion element
IS6110. The first step of RFLP is purification of DNA from an M.
tuberculosis isolate. A restriction enzyme is added that cuts the
DNA into hundreds of different fragments at specific sequences. The
fragments are separated by size on an agarose gel and transferred to
a membrane. A probe is then used to detect fragments containing
IS6110, and the image is captured on film. Each copy of IS6110
produces one band. IS6110-based RFLP patterns containing 7 or more
bands provide more specificity in discriminating between isolates
than do patterns with 6 or fewer bands.
What is a TB genotype cluster?
When two or more M. tuberculosis
isolates match by genotyping
methods (i.e., same spoligotype and MIRU patterns), they are
referred to as a genotype cluster. Patients who are members of the
same genotype cluster are assumed to have the same strain, which may
be a surrogate for recent transmission. However, genotyping
information is only one piece of evidence used to determine
transmission patterns. Genotyping information, epidemiologic
linkages including spatial (geography) and temporal (time)
associations, and drug susceptibility results (phenotype) can help
distinguish recent transmission from activation of latent TB
NTGS laboratories assign unique numbers to clusters specifying
each spoligotype and MIRU pattern combination. For each combination,
a national and a state-specific designation are assigned. For
example, all isolates within the United States with an identical
spoligotype pattern such as 777776777760601 and MIRU pattern
224325153323 are assigned a national designation such as PCR00015.
State-specific designations are also used for the same pattern. For
example, in Arkansas, the state-specific designation for this
pattern could be AR_010; whereas, in California it could be CA_084;
in Colorado it could be CO_016; and in Nevada it could be NV_016.
The national designations are designed to help facilitate
inter-jurisdictional communication between TB controllers so they
don’t have to report 15- and 12- digit numbers; whereas, the state
designations provide ease of use within the jurisdiction.
Click here for a
detailed description of cluster designations
How can TB genotyping information help identify unsuspected
Patients with TB disease caused by the same strain of M.
tuberculosis will have matching genotype results. If an
investigation establishes that two or more TB patients with matching
genotypes share known epidemiologic linkages, this provides good
evidence that these patients were involved in the same chain of
However, typically 20%–40% of genotype-matched TB cases are not
identified as being connected by contact investigation. During
traditional contact investigations, public health workers interview
TB patients to elicit the names of other people who may have been
exposed to them, and venues in which exposure may have taken place.
TB patients are often unable or reluctant to name all contacts and
all places of potential transmission. A careful review of public
health records, genotyping results, contact investigation logs,
estimated infectious periods, and re-interview of patients in a
genotype cluster can uncover additional potential transmission
How can TB genotyping help in source-case investigations for
pediatric TB cases?
Pediatric cases are considered sentinel events in that they
provide evidence of recent TB transmission. Source-case
investigations can potentially identify additional TB cases, confirm
suspected transmission links between a suspected source case and the
child, or identify new, previously unsuspected, venues of
transmission. When a suspected source case is identified, genotyping
can support evidence of the transmission link to the child if the
isolates from both have matching genotypes. If the investigation
yields no suspected source case, a review of all genotyping results
from patients living in the same region as the child may identify a
genotype match. Also, further interviews of patients with matching
genotypes may reveal the true source for the child’s TB.
In patients with multiple episodes of TB, how will TB genotyping
help distinguish between reactivation of an old infection or recent
infection with a new strain?
Reactivation occurs when a patient with TB is treated and cured,
but has a subsequent episode of TB disease caused by the same strain
of M. tuberculosis
as the previous episode. Reinfection (with
development of disease) is caused by a second infection with a
strain that usually has a different genotype from the strain that
caused the initial episode of TB disease. Genotyping the initial
isolate and the subsequent isolate from the same patient can
distinguish between these two possibilities.
How can TB genotyping find or confirm false-positive cultures?
An estimated 2% of all M. tuberculosis
false-positive results. This can occur in even the most proficient
laboratories for a variety of reasons (e.g., mislabeling,
cross-contamination). A false-positive culture should be suspected
when patients have a single culture confirmation, clinical
presentation inconsistent with TB, or no clinical improvement
despite adequate TB therapy. Public health officials must work
closely with laboratorians and clinicians to gather information to
verify or refute that suspicion.
A false-positive culture investigation is a multistep process
that requires an investigation of the entire path a specimen and
isolate take, from collection through the final laboratory report.
This process enables the identification of possible common
collection or processing points, which, at a given point in time,
could have resulted in cross-contamination or mislabeling.
What is universal TB genotyping? Are we there yet?
Universal TB genotyping means submitting to the genotyping
laboratories at least one isolate for every culture-confirmed TB
case in the country. Universal TB genotyping will provide the best
understanding of the epidemiology of TB transmission within a
specific TB program’s jurisdiction, as well as the entire country.
It may uncover the greatest number of unrecognized outbreaks,
clusters, and false-positive cultures. In 2004, when the NTGS was
initiated, only 47% of all culture-positive TB cases in the United
States had genotyping information. In 2007, this proportion had
increased to 86%.
How can I receive and manage TB genotyping data for my patients?
The TB Genotyping Information Management System (TB-GIMS) will be
launched in early 2009 to track and manage M. tuberculosis
genotyping data. TB-GIMS links genotyping results to the
epidemiologic data from surveillance reports; queries and reports
can be generated for comparing genotyping results locally and
nationally. In the future, TB-GIMS will generate alerts or
notifications of suspected recent transmission and help identify TB
clusters that suggest public health action.
Click here for a
description of TB-GIMS.
How much does TB genotyping cost patients or health care
TB genotyping is provided at no cost to patients, healthcare
providers, and health departments. CDC also pays for shipping of
isolates for genotyping from local laboratories to the genotyping
laboratories. Shipping labels are provided from CDC to facilitate
the shipping process.
For more information on TB genotyping, please contact