Guide to the Application of Genotyping to Tuberculosis Prevention and Control

CDC Tuberculosis Genotyping Laboratory Procedures

Submission of Isolates

TB Genotyping Isolate Submission Form

The submitting laboratory will be responsible for completing the TB Genotyping Isolate Submission Form when they submit isolates to the genotyping laboratory. An electronic copy of this Excel spreadsheet form is available for downloading from the TB WebBoard at http://web-tb.forum.cdc.gov.

It is absolutely critical that TB programs and laboratories NOT change in any way the column headings of the Isolate Submission form spreadsheet. Any change in the column headings will result in data being lost.

The spreadsheet contains three optional columns (the column headings are “option_1”, “option_2” and “option_3”), which the TB programs can use to record any information they want, but they must not change the column headings for these fields either. For example, the column labeled as “option_1” could be used to record the zip code of patients, but the heading must remain “option_1” on the submitted form. Any optional columns that the program does not want to use can be deleted, and the order of the columns on the form can be changed to suit the program.

Guidelines for Submission

The TB Genotyping Isolate Submission Form is an Excel spreadsheet, and laboratories that can use Excel should enter the isolate information directly onto the spreadsheet. One spreadsheet line should be used for each isolate. List all of the isolates that will be shipped in one container on one spreadsheet; print and insert a copy of the spreadsheet into the container. In addition to mailing a hard copy of the spreadsheet with the isolates, send a copy of the Excel spreadsheet as an e-mail attachment to the genotyping laboratory. Send a courtesy copy to the state TB program for matching with the TB program’s list of new cases and with later genotyping results. Patient confidentiality must be protected, but sufficient patient identification data are needed to allow this reconciliation to occur without error.

Special procedures will be required for submitting laboratories that do not have access to Excel. They should

• enter the required information onto a sheet of paper, using a separate sheet for each patient and
• mail these pages and a packing list of all the isolate identification numbers with the isolates.

Submitting laboratories that can use Excel but cannot e-mail Excel attachments should print out hard copies of the Excel spreadsheet and mail them to the genotyping laboratory.

Upon receipt of the isolates, the genotyping laboratory will match the isolates that arrive in the shipment with the printed copy of the spreadsheet that accompanies the shipment. If all of the isolates are accounted for, the genotyping laboratory will acknowledge receipt to the submitting laboratory by e-mail; a courtesy copy will be sent to the TB program. For submitting laboratories that cannot receive acknowledgement by e-mail, the genotyping laboratory will mail a hard copy acknowledgement. Isolates will be assigned genotyping laboratory accession numbers that will include the year, consecutive numbers, and a letter code that represents the genotyping laboratory identification designation.

Material for Isolate Submission

Because only small amounts of culture material are required for the PCR-based typing methods, submitting laboratories should prepare an isolate for shipment as soon as it is identified as M. tuberculosis complex (including M. bovis and Mycobacterium africanum). Portions of the culture material should be placed in 2-ml screw-cap vials, which will provide substantial savings in shipping costs. Although the process of subculturing onto Lowenstein-Jensen (L-J) medium (waiting 3 weeks for visible growth and then shipping the L-J slant) is acceptable, it greatly delays turnaround times and substantially increases shipping costs.

The genotyping laboratory will subculture submitted material for possible IS6110-based RFLP typing and long-term storage. Isolates that do not grow on subculture at the genotyping laboratory will be typed by spoligotyping and MIRU, and replacements for these non-viable isolates will be requested from the submitting laboratory.

Acceptable Material for Isolate Submission

• Portions of culture material from solid medium transferred to 2-ml screw-cap vials
• Samples (0.5-1.0 ml) of broth culture transferred to vials
• Portions (0.5-1.0 ml) of broth cultures or suspensions prepared for susceptibility testing
• Suspensions (0.5-1.0 ml) prepared for Accuprobe testing (before lysis)
• L-J (or other solid medium) slants or bottles (not optimal; see discussion in the previous section, Material for Isolate Submission

Unacceptable Material for Isolate Submission

• Clinical specimens (e.g., sputum, pleural fluid)
• Sediments from processed clinical samples, regardless of smear or nucleic acid amplification positivity
• Petri plates
• Cultures not yet identified as M. tuberculosis complex
• Obviously contaminated or suspected mixed cultures

Shipping Guidelines

• Each sample must be labeled clearly with a patient identifier, and the same patient identifier must be listed on the TB Genotyping Isolate Submission Form.
• Liquid samples should be shipped in plastic screw-cap tubes with o-rings. The tubes should be surrounded by absorbent material.
• Containers and labels must comply with all regulations of the U.S. Department of Transportation and the International Air Transport Association for shipment of infectious material.
• Shipping personnel should have completed required training.
• See Information on Packaging and Shipping Infectious Substances in Appendix B, under Useful Resources, for additional information.