The primary objective of this study was to compare two amplification techniques in their ability to detect M. tuberculosis
in respiratory specimens. Culture of M. tuberculosis
in a liquid medium and auramine staining were the reference methods (2
At the Erasmus MC site, the COBAS AMPLICOR MTB technique is routinely applied together with the MGIT as a liquid culture technique. At the other collaborating center, the BDProbeTec ET technique and the BACTEC liquid culture technique are applied together in the routine setting. After specimens had been processed for culture and microscopy, subsamples of the specimens were exchanged between the participating centers to be analyzed with the respective amplification methods (2
Although the specimen volume was corrected, different results were obtained using the two amplification systems, as the COBAS AMPLICOR MTB system detected 85/109 (78%) and BDProbeTec ET system detected 94/109 (86.2%) culture-positive specimens. The majority of the differences found between the two systems came from the auramine-negative specimens. In 42 auramine-negative but culture-positive specimens, BDProbeTec ET detected 27 (64.3%) and the COBAS AMPLICOR MTB detected 21 (50%) positive specimens. The limited value of amplification assays in detecting true positives in auramine-negative specimens has been reported earlier by Piersimoni et al. (19
Our results were comparable with the results obtained by Ichayama et al. (9
), who reported sensitivities of 89.5% and 94.7% for COBAS AMPLICOR MTB and BDProbeTec ET, respectively. Similar results were obtained by Kim et al. (12
); however, interpretation of these data is difficult, as only a limited number (n
= 26) of positive specimens were studied. Iinuma (10
) did not find significant differences between the two amplification methods, but this is due to the fact that the specimens evaluated were almost all auramine positive. From our data, it is shown that there is no difference between the two amplification methods for the auramine-positive specimens. The differences between the amplification methods are observed with the auramine-negative specimens.
The COBAS AMPLICOR MTB and BDProbeTec ET systems detected additional positive specimens, though after studying the medical history of the patients involved, we found that most of these additionally positive specimens came from patients who were undergoing treatment or who had been previously treated for M. tuberculosis
during the last 6 months. Hellyer et al., Levee et al., and Thomsen et al. (8
) previously demonstrated that M. tuberculosis
DNA could be detected by strand displacement amplification and PCR in frozen sputum samples more than 12 months after the initiation of treatment and more than 6 months after positive-to-negative culture conversion.
The differences found between the two amplification systems could be explained by differences in the extraction methods used or by a reduction in the amount of inhibitory factors. The difference in sensitivity cannot be explained by differences in the volume used in the two amplification reactions. In the COBAS AMPLICOR MTB system, 25 μl of the primary extract is actually used in the amplification reaction. In the BDProbeTec ET assay, 13.75 μl is used. However, when the input volume is reduced, the input of inhibitory substances is also reduced, and the difference in sensitivities is therefore probably due to a better extraction method in combination with a reduction in inhibition, leading to optimized amplification. This presumably explains the absence of inhibited samples for the BDProbeTec ET assay in our study. In the COBAS AMPLICOR MTB system, seven specimens (0.85%) were reported to be inhibited, which leads to inconclusive results. Inhibition rates for the COBAS AMPLICOR MTB varying from 0.3% to 3.9% for respiratory and nonrespiratory specimens have been reported by others (20
). In contrast to these findings, Kim et al. (12
) reported an inhibition rate of 0.7% for BDProbeTec ET and of 0% for COBAS AMPLICOR. As the inhibition rates vary considerably, we suggest that the differences found between the two methods are probably due to a better extraction protocol applied by the BDProbeTec ET technique. Auramine-negative specimens have a low number of bacteria, and with these specimens, differences between results of the two methods are observed. Probably, due to the mechanical disruption applied by the BDProbeTec ET technique, more specific DNA of the organisms is extracted and becomes available for amplification.
At present, liquid culture techniques still appear to be superior to the amplification techniques in the diagnosis of M. tuberculosis infection. The difference in sensitivities between culture and molecular (standard PCR) testing is most probably due to the differences in input volume; culture is better due to a larger inoculum size. In our study, the advantage of the molecular techniques is lost for the auramine-negative group, i.e., the specimens with a low number of acid-fast bacilli.
From our results, the COBAS AMPLICOR MTB and BDProbeTec ET techniques have an added value for specimens that are also found to be auramine positive. In this respect, the COBAS AMPLICOR MTB and BDProbeTec ET amplification systems easily discriminate between M. tuberculosis and non-M. tuberculosis isolates, giving relevant information to the clinician with respect to diagnosis and optimal treatment of a particular patient. Both COBAS AMPLICOR MTB and BDProbeTec ET systems have an acceptable sensitivity and an excellent specificity. A difference in sensitivities between the results obtained for the present study and the results presented in previous publications may be attributed to the use of different reference methods, the use of different patient specimens (i.e., tissue specimens), or the inclusion or exclusion of auramine-negative specimens.
In conclusion, the sensitivities of the BDProbeTec ET system and the COBAS AMPLICOR MTB appear adequate for the analysis of auramine-positive samples. For auramine-negative samples, however, the number of false-negative results is still too high. Our results confirm the prevailing opinion that amplification techniques should currently serve only as an adjunct to culture and certainly not as a replacement.