TB Elimination

October 2008 Website - www.cdc.gov/tb Page 1 of 3

Tuberculosis Genotyping

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 patients

 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 outbreak

 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 nontuberculous 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 isolates: spacer oligonucleotide typing

(spoligotyping) and variable-number tandem repeat of

mycobacterial interspersed repetitive units (VNTRMIRU). These methods yield digital resultsthat can be

readily analyzed. A detailed description of these methods is available at: http://www.cdc.gov/tb/genotyping/

Chap3/3_CDCLab_2Description.htm

 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).
October 2008 Website - www.cdc.gov/tb Page 2 of 3

 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. Details on when to request RFLP

is available at: http://www.cdc.gov/tb/genotyping/

Chap5/5_Developing_3c_RFLP.htm

 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 infection.

NTGS laboratories assign unique numbers to clusters

specifying each spoligotype and MIRU pattern combination. For each combination, a national and a statespecific 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

A detailed description of cluster designations is available

at: http://www.cdc.gov/tb/genotyping/Chap3/

3_CDCLab_4Results.htm

How can TB genotyping information help

identify unsuspected epidemiologic links?

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 recent

transmission.

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 opportunities.
How can TB genotyping help in sourcecase 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 cultures

represent 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 culturepositive 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 isolate genotyping data. TBGIMS 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. A more detailed description of TB-GIMS is

available at: http://www.cdc.gov/tb/pubs/tbfactsheets/

gims.htm.

How much does TB genotyping cost

patients or health care providers?

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.

Additional Information

For more information on TB genotyping, please contact

tbgenotyping@cdc.gov

Safer . Healthier Healthier . People

October 2008 Website - www.cdc.gov/tb Page 3 of 3