Abstract:
Bovine tuberculosis is a global cause for concern in livestock, free-ranging wildlife,
zoological collections and the human population. Large amount of time, effort and
resources are spent on its diagnosis and control methods. This study was aimed at
determining the sensitivity and specificity of the IS6110 specific PCR test on formalin
fixed, paraffin embedded (FFPE) tissue blocks, compared to that of the gold
standard method culture and to differentiate M. bovis from other members of the M.
tuberculosis complex using the RD4 region of difference specific PCR test. A total of
141 FFPE tissue blocks of wild animals from game reserves, the National Zoological
Gardens and routine tuberculosis (TB) surveys in Kruger National Park were tested.
Among the 50 known TB positive samples (35 M. bovis culture positive, twelve M.
tuberculosis culture positive and three diagnosed tuberculosis positive on
histopathology examination) the IS6110 PCR had an overall sensitivity of 22%. The
positive predictive value of the IS6110 test (91.67%) was quite high implying that
although sensitivity was low, one can be highly confident that a positive test result is
a true reflection of the positive disease status. The overall sensitivity of the RD4 PCR
was 20%. The positive predictive value of the RD4 test (41.67%) was low, implying
that a positive test result may be unreliable. The sensitivities of the M. tuberculosis
and M. bovis culture positive samples were compared and a significant difference
was noted. Sensitivities of the IS6110 and RD4 assays in M. tuberculosis culture
positive samples were 66.67% and 33.33%, respectively; sensitivities of the IS6110
and RD4 assays in M. bovis culture positive samples were 8.57% and 17.14%,
respectively. Difference in bacterial load in tissues infected with the two
mycobacterial species may account for this finding (i.e. M. bovis infections have a
lower bacteria load). Of the 91 known TB negative samples, the specificity of the
IS6110 (98.90%) and RD4 (84.62%) PCR tests were high, but the negative
predictive values of 69.67% and 65.81%, respectively, suggest that the probability of
negative test results being incorrect still exists. The resultant sensitivity was
increased when parallel interpretation was applied to histopathology examination
and the IS6110 or RD4 PCR tests and when applied to the IS6110 and RD4 PCR
tests. Both histopathology examination and PCR tests produce rapid results and
their combination can be used in routine diagnostics. The RD4 PCR assay was
unable to distinguish M. bovis from other members of the MTB complex and based
on the findings of this study the RD4 PCR cannot add value to the diagnosis of
suspect tuberculosis samples at this stage, but successful troubleshooting relating to
1) extraction method, 2) DNA inhibitors, 3) contamination and 4) multisampling protocol, may enable its use in future.
