Guide to the Application of Genotyping to Tuberculosis Prevention
Developing a Tuberculosis Genotyping Program
Establishing Procedures for Requesting RFLP Analysis
The two PCR-based genotyping methods (spoligotyping and MIRU analysis)
provide sufficient discriminatory power for most TB control purposes.
IS6110-based RFLP analysis can provide additional discriminatory
power, but usually it is not needed. Because RFLP analysis is labor-intensive,
its use should be limited to situations where additional information
that can be provided only by RFLP will guide further action.
RFLP analysis is indicated only for isolates that have already
been found to have matching PCR genotypes. One purpose of genotyping
is to determine whether isolates are genetically distinct and therefore
unlikely to be involved in the same chain of recent transmission.
If any genotyping test shows that two isolates have different genotypes,
this provides strong evidence that the two isolates are genetically
distinct. Additional testing of genetically distinct isolates will
not change the conclusion that they are genetically distinct. Therefore,
if two isolates have different spoligotypes or different MIRU types,
the results of the RFLP analysis will rarely be helpful. In the
following discussion of when to request an RFLP analysis, it is
assumed that isolates have already been tested by spoligotyping
and MIRU analysis and that both tests resulted in matches.
TB programs should request RFLP analysis usually for isolates from
patients who are involved in ongoing investigations of outbreaks,
because it is important to know with as much accuracy as possible
whether the isolates represent the same strain of M. tuberculosis.
During outbreak investigations and during contact investigations
of multiple cases with isolates that match by the two PCR tests,
RFLP results help to establish which patients belong to the outbreak
and which patients do not. Either result helps to focus the outbreak
investigation on those patients involved in the same chain of transmission.
Some false-positive culture investigations should include RFLP
analysis, but others do not require it. The determination of when
to request RFLP analysis during a false-positive culture investigation
rests on whether there was a preexisting suspicion of a problem.
In circumstances where the treating physician or the clinical laboratory
suspects a false-positive culture, a match by spoligotype and MIRU
analysis is sufficient to confirm that suspicion. On the other hand,
if the PCR genotyping results showing a possible false-positive
culture were unexpected by the laboratory and the clinicians, RFLP
analysis should be requested to confirm the PCR results.
When patients who are clustered by the two PCR tests are thought
to be involved in the same chain of recent transmission and previous
contact investigations established known epidemiologic links between
them, chances are very good that RFLP results will confirm the genetic
identity of the isolates. Therefore, in this case, RFLP analysis
is not indicated. In contrast, for similar patients for whom only
possible epidemiologic links have been found (i.e., PCR test results
match and patients could be involved in recent transmission), RFLP
analysis should be considered. Here, the presence of possible epidemiologic
links raises the question of whether the patients represent the
same chain of recent transmission. If RFLP results show that the
isolates are different, the TB program will decide that no cluster
investigation is needed because the RFLP results indicated that
no cluster existed. If the RFLP pattern shows a match, the TB program
will have added motivation to conduct a cluster investigation.
A final situation that could warrant a request for RFLP analysis
occurs when certain strains of M. tuberculosis match by spoligotyping
and MIRU analysis but can be differentiated only by RFLP. This situation
may become increasingly important as we gain more information about
the distribution of isolates that match by spoligotyping and MIRU
analysis. The genotyping laboratories will be a good source of information
about the need for conducting RFLP analysis in this situation.
When to Request IS6110-based RFLP
Request RFLP only for isolates that have matching spoligotyping
and MIRU genotypes and when the RFLP results may alter your
decision about further action steps. If these two prerequisites
are met, use the following criteria to help you make a decision.
Usually request RFLP when conducting
- an outbreak investigation
- certain false-positive culture investigations
Consider requesting RFLP when investigating
- clustered patients with possible epidemiologic links
Consult with the genotyping laboratory when investigating
strains that are known to cluster by the PCR tests but can
be distinguished by RFLP.