Appendix C

Types of data

Tuberculosis (TB) genotyping is a laboratory-based approach used to analyze the genetic material (i.e., DNA) of Mycobacterium tuberculosis, the bacterium that causes 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.

Conventional TB genotyping examines the location, number, and presence of different types of spacer or repetitive DNA patterns. The areas of the genome examined in TB genotyping are different from those related to drug resistance. Whole-genome sequencing (WGS)-based TB genotyping examines 70%–90% of the bacterial genome.

How information is collected

In 1996, the Centers for Disease Control and Prevention (CDC) started the National Tuberculosis Genotyping Surveillance Network (NTGSN). NTGSN was a 5-year initiative that established the utility of genotyping in TB control efforts1.

In 2004, using knowledge gained from NTGSN and associated studies2, CDC established the National Tuberculosis Genotyping Service (NTGS). CDC funded national genotyping laboratories to genotype ≥1 M. tuberculosis isolate from each culture-positive TB case reported in the United States3. All U.S. TB control programs can use NTGS at no cost to patients, health care providers, or health departments. NTGS participation is voluntary, with each program determining how to use genotyping data for its TB control activities. Since 2004, NTGS and its partners genotyped approximately 180,000 M. tuberculosis isolates.

In 2010, CDC launched the Tuberculosis Genotyping Information Management System (TB GIMS), a secure web-based application available to reporting areas including:

• All 50 U.S. states,
• District of Columbia,
• Puerto Rico,
• U.S. Virgin Islands, and
• U.S.-affiliated Pacific Islands.

TB GIMS makes genotyping data available to users. It also facilitates linking TB genotyping data to patient surveillance records from the National TB Surveillance System. Key features of TB GIMS include:

• Queries of a database of genotypes and clusters,
• Data quality checks,
• Aggregate reports, and
• Outbreak detection tools.

As of September 2024, TB GIMS has 623 users among state, tribal, local, territorial, and federal partners.

In 2018, CDC established the National Tuberculosis Molecular Surveillance Center (NTMSC). NTMSC performs whole-genome sequencing (WGS) on ≥1 isolate from every culture-positive TB case in the United States. In July 2022, NTMSC transitioned from conventional genotyping (e.g., GENType) to WGS-based genotyping and the resulting whole-genome multilocus sequence type (i.e., wgMLSType). However, existing data from conventional genotyping remains available in TB GIMS for jurisdictions to reference for TB prevention and control activities.

How the data are used

When recent TB transmission occurs, the persons with TB disease often have a matching TB genotype. Likewise, persons who have a matching genotype are more likely to be related by transmission than persons without a matching genotype. The connection might be due to recent TB transmission or a previously acquired TB infection in the remote past.

Genotyping results, when combined with epidemiologic data, can help public health experts identify persons with TB disease involved in the same chain of transmission. This information adds value to TB control activities in a number of ways.

Person-level applications of genotyping

Contact investigations

Using genotyping in contact investigations can help support or refute connections between ≥2 persons with TB disease (i.e., epidemiologic linkages). Public health experts might or might not have established connections between the persons during contact investigations.

Cluster investigations

In cluster investigations, genotyping can suggest connections that contact investigations did not establish. Public health experts should investigate these connections further.

Other applications

• Distinguish relapse TB disease from new TB infection (i.e., reinfection) among persons with recurrent TB disease.
• Detect and investigate potential false-positive culture results.

Population-level applications of genotyping

Outbreak detection, assessment, and monitoring

• Identify genotype clusters that could represent an outbreak using geospatial or other analyses.
• Assess the genetic relatedness of isolates among cases believed to be part of an outbreak.
• Define the scope of potential outbreaks by identifying all presumed cases in an area with a matching genotype.
• Monitor outbreaks for new cases.

Genotyping-based cluster detection

CDC identifies genotype-matched clusters using geospatial analysis to identify unexpected clustering of TB cases within a defined time period. CDC bases its primary cluster detection method on identifying higher than expected geospatial concentrations of a TB genotype in a specific county compared with the national distribution of that genotype over a 3-year period. This cluster detection method calculates a log-likelihood ratio (LLR) statistic. Clusters with higher LLRs represent greater geospatial concentrations than clusters with lower LLRs. Higher LLRs might indicate recent transmission of TB. TB GIMS classifies clusters into alert levels based on established LLR cut points:

• No alert (LLRs 0–<4),
• Medium alert (LLRs 4–<10), or
• High alert (LLRs ≥10).

The alert level and changes in alert levels can help TB programs prioritize TB genotype clusters for further investigation and possible intervention. This can help to identify outbreaks of TB disease. CDC assesses characteristics of alerted clusters and communicates findings for priority clusters back to TB programs.

National TB genotyping surveillance coverage in the United States

Genotyping surveillance coverage refers to the percentage of culture-positive TB cases with a genotyped M. tuberculosis isolate. High levels of coverage can provide a better understanding of the molecular epidemiology of TB transmission within a specific geographic area and nationally.

Outbreak detection algorithms identify unexpected geospatial concentrations of persons who have isolates with the same genotype. Therefore, high coverage levels help decrease the likelihood of failing to alert clusters of TB disease that may be of concern. The genotyping surveillance coverage was 96.0% in 2023; the National Tuberculosis Indicators Project target for the United States was 100% for 2023.

Genotyping terminology

From 2009 to 2022, NTGS defined a genotype as a unique combination of:

• Spacer oligonucleotide typing (spoligotype) and
• 24-locus mycobacterial interspersed repetitive unit–variable number tandem repeat typing (MIRU–VNTR) results.

Each unique combination of results was assigned a GENType designated as G followed by 5 digits. The 5 digits were assigned sequentially to every genotype identified in the United States (e.g., G00162).

Since 2018, genotyping has included WGS-based methods that compare about 70% of the M. tuberculosis genome. Nationally, isolates with ≥99.7% of their loci (i.e., genes) matching are considered clustered. They are sequentially assigned a wgMLSType designated by MTBC followed by 6-digits (e.g., MTBC000123). Otherwise, the isolate is designated as "MTBCunique."

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