Guide to the Application of Genotyping to Tuberculosis Prevention
Tuberculosis Genotyping Case Studies: How TB Programs
Have Used Genotyping
False-Positive Culture Investigation
In early 2003, a state mycobacteriology laboratory began testing
the Mycobacterium Growth Indicator Tube (MGIT) 960 system as a potential
replacement for their current BACTEC 460 system for culture of M.
tuberculosis in broth media. The MGIT 960 is an automated culture
system that automatically checks culture tubes for growth every
hour and does not depend on a technician reviewing culture results
twice each week, as was required by the former system. During February
and March 2003, the BACTEC and the MGIT systems were used in parallel.
In March 2003, several M. tuberculosis isolates cultured
in the state laboratory were reported to be resistant to isoniazid
(INH) and streptomycin. In April 2003, several isolates also demonstrated
resistance to isoniazid (INH) and ethambutol. Since these susceptibility
patterns are unusual, the concern for possible false-positive culture
results was raised and a formal investigation was initiated.
Spoligotyping and MIRU analysis identified three separate genotyping
clusters during this time period. The first cluster consisted of
two isolates with isoniazid and streptomycin resistance. One of
the isolates came from a patient with three positive cultures and
an abnormal chest x-ray; the other came from a patient with only
one positive culture and a history of blunt trauma to the chest.
This patientís specimen was processed 1 day after the first patientís
specimen was processed for drug susceptibility testing.
The second cluster involved isolates from six patients, each with
only one positive culture. Only one patient had a clinical picture
suggestive of tuberculosis, and this patient was in his 11th
month of anti-TB treatment administered by directly observed therapy.
The spoligotyping and MIRU results from these six isolates matched
a quality-control strain used by the laboratory. In each instance,
patient specimens were processed for drug susceptibility testing
within 1-2 days of the quality-control strain.
The third cluster involved only one patient. This patient had only
one positive culture result and had a clinical picture that was
not particularly suggestive of tuberculosis disease. This patientís
isolate matched the genotyping pattern of a proficiency strain the
laboratory processed 1 day before during drug susceptibility testing.
A review of laboratory procedures revealed that new laboratory
protocols were required for use of the MGIT system. These protocols
stipulated that a manual micropipettor should be used to inoculate
tubes for drug susceptibility testing and for adding sterile supplement
to broth tubes for culture of new specimens. Additional micropipettors
had been ordered, but they had not arrived yet at the time of the
contamination. Therefore, the same manual micropipettor was used
to inoculate tubes for DST each afternoon and to add sterile supplement
to broth tubes each morning. These broth tubes were subsequently
inoculated with incoming specimens/isolates via sterile, disposable
This investigation highlights several common findings when false-positive
cultures occur. All of the patients with false-positive cultures
had only one positive culture result (all the true cases had more
than one positive culture result). All of the patients with false-positive
culture results did not have clinical pictures that were particularly
suggestive of active tuberculosis, and in all cases, the contaminated
cultures and the cultures that were the sources of the contamination
were processed within 1-2 days of each other. Genotyping helped
the laboratory staff and clinicians communicate rapidly and terminate
As you will see when we discuss algorithms for identifying possible
false-positive culture results, each of these findings are red flags
and should be evaluated each time a genotyping cluster is reported.