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Guide to the Application of Genotyping to Tuberculosis Prevention and Control

CDC Tuberculosis Genotyping Laboratory Procedures

Genotyping Results, Designation of Clusters, and Reporting

Turnaround Times of Genotyping Results

CDC’s contract with the genotyping laboratories requires that the laboratories genotype all of the eligible isolates by PCR tests (spoligotyping and MIRU analysis) and report results for at least 90% of isolates to the TB programs within 10 working days of receipt of the isolates. These rapid turnaround times assume a steady flow of isolates to the laboratory. Although we expect that these projected turnaround times will be met most of the time, there are several reasons for possible delays. The MIRU automated sequencer is limited to 24 isolates in each run; if a genotyping laboratory receives large batches at one time, there will be a delay in reporting some of the results. Technical difficulties (e.g., instrument failure) may also cause delays.

Under certain circumstances, the TB program can request that the genotyping laboratories perform IS6110-based RFLP analysis on isolates that match by both spoligotyping and MIRU analysis. See “IS6110-based RFLP Analysis” (Chapter 3, CDC Tuberculosis Genotyping Laboratory Procedures) for details about how the genotyping laboratory will report RFLP results, and see chapter 5, Developing a Tuberculosis Genotyping Program, for a description of circumstances where RFLP analysis is appropriate. CDC’s contract stipulates that the genotyping laboratories will report RFLP results for 90% of isolates within 15 working days from the date of the RFLP analysis request.

In almost all instances, these turnaround times are fast enough to ensure that there will be no need for TB programs to prioritize requests, even for investigations of outbreaks or false-positive cultures.

Designation of Clusters and Reporting

Automated instruments determine the spoligotype and MIRU type. The results (15-digit octal number for spoligotypes and 12-digit number for MIRU types) are imported directly into the genotyping laboratory-tracking database. The automated nature of the analyses and assignment of numeric results reduces human interpretation and transcription errors.

The spoligotype and MIRU type will be compared with all other isolates previously submitted from the same TB program. The genotyping laboratory will assign a PCR cluster designation to isolates that have spoligotypes and MIRU types that match exactly. The PCR cluster designation will consist of a two-letter TB program designation followed by a consecutively derived three-digit number. For state programs, the two-letter designation will be the postal code (e.g., “GA” for Georgia’s state TB program). See Chapter 4, Combining Genotyping and Epidemiologic Data to Improve Our Understanding of Tuberculosis Transmission, for examples.

For each new isolate from a specific TB program, the search for matches will yield one of three results:

  • No match. This indicates that a search of current and previous isolates from the TB program identified no other isolate with a matching PCR genotype. Because the search for matches will be restricted to isolates from the TB program, it is possible that a matching isolate from another TB program exists in the genotyping laboratory’s database. See Chapter 5, Developing a Tuberculosis Genotyping Program, for a discussion of how TB programs can compare genotyping results with other TB programs.
  • A match with one current or previous isolate. The genotyping laboratory will assign a new genotyping cluster number to this newly identified cluster.
  • A match with more than one isolate from an existing genotyping cluster. The genotyping laboratory will assign the existing cluster number to this newly identified addition to an existing cluster.

When this matching analysis is complete, the results will be sent by e-mail or other means to the TB program. At a minimum, the report will list for each isolate the original isolate number supplied by the submitting laboratory, the accession number assigned by the genotyping laboratory, the report date, the spoligotype and MIRU type, and cluster designation (for clustered isolates). For new clusters, the report will list the previous isolate that matches the new isolate so that the TB program can update their database. These reports will be generated automatically, and each program can specify any other data from the original submission form to be included in the report. It is the responsibility of the TB program to distribute the genotyping results as they see fit to others in their jurisdiction.

 
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