Comparison of Roche Cobas Amplicor Mycobacterium tuberculosis Assay with In-House PCR and Culture for Detection of M. tuberculosis

Abstract

The new Roche Cobas Amplicor Mycobacterium tuberculosisassay, which is a semiautomated version of the manually performed Roche Amplicor M. tuberculosis test, was compared to culture and an IS6110-based in-house PCR protocol. A total of 1,681 specimens from 833 patients, including specimen types other than sputum, were tested in parallel by both the in-house PCR and the Cobas Amplicor M. tuberculosis assay. After we resolved discrepant PCR results, the sensitivity, specificity, and positive and negative predictive values for the Cobas Amplicor M. tuberculosis assay were 66.33, 99.71, 94.36, and 97.66%, respectively. The corresponding values for the in-house PCR were 91.08, 99.85, 97.87, and 99.37%, respectively. For culture- and smear-positive specimens, the sensitivity of the Cobas AmplicorM. tuberculosis test was 96.42% (in-house PCR, 100%). If only smear-negative sputum specimens were considered, the Cobas Amplicor M. tuberculosis assay exhibited a sensitivity of 45.45% (in-house PCR, 63.63%) relative to that of culture. With a modified protocol for DNA extraction (washing of samples plus ultrasonication), both PCR methods performed better with gastric aspirates than with sputum samples (sensitivity of the Cobas AmplicorM. tuberculosis assay with smear-negative gastric aspirates, 70.00%; sensitivity of in-house PCR, 90.00%). With dithiothreitol being used for liquefaction of specimens in this study, the Cobas Amplicor M. tuberculosis assay exhibited an inhibition rate of 9.16%. In our view, the new Cobas Amplicor M. tuberculosis test (i) is well suited for typing of smear-positive specimens, (ii) may also be applied to gastric aspirates and other types of specimens if DNA extraction methods are modified appropriately, and (iii) exhibits a sensitivity with smear-negative sputum specimens which makes it recommendable that a minimum of three samples from the same patient be tested.

Comparison of Enhanced Mycobacterium tuberculosis Amplified Direct Test with COBAS AMPLICOR Mycobacterium tuberculosis Assay for Direct Detection of Mycobacterium tuberculosis Complex in Respiratory and Extrapulmonary Specimens

“…In fact, most of these samples (13 of 16), which in a routine setting were likely to be misdiagnosed to contain NTM or considered as negative, turned out positive by simple dilution. All TB positive (41) 34 [4] 34 ( A considerable increase in the specimen volume (from 50 to 450 l) in comparison with the former version has been claimed as the most likely explanation of this previously undescribed inhibition (5).…”

“…Mycobacteriol., p. 76, 1999). However, because the nature of the inhibition is still unclear and probably affects amplification techniques unevenly, it seems unlikely we will be able to find a routine-fitting procedure able to remove all inhibitory substances (3,4).…”

Automatized PCR evaluation ofMycobacterium tuberculosiscomplex in respiratory and nonrespiratory specimens

“…In a number of studies it was stressed that testing with multiple specimens from one patient had increased the sensitivity of the COBAS AMPLICOR MTB PCR assay in smear-negative tuberculosis (Wobeser et al, 1996;Bodmer et al, 1997;Yuen et al, 1997;Rajalahti et al, 1998;Lim et al, 2000;Johnsson & Ridell, 2003;Levidiotou et al, 2003;Kim et al, 2004). By contrast, in smear-positive specimens the rapid diagnosis and/or confirmation of the clinical diagnosis and differentiation between M. tuberculosis and nontuberculous mycobacteria is emphasized (Yuen et al, 1997;Eing et al, 1998).…”

Molecular genetic methods for diagnosis and antibiotic resistance detection of mycobacteria from clinical specimens

“…In another comparative study of the above two tests in a total of 1, 681 clinical specimens, the Cobas Amplicor MTB test was again found to have a lower sensitivity of 66.33% compared to 91.08% for the ‘in‐house’ IS 6110 ‐PCR. The specificities were comparable (44). Observed differences in sensitivity between the Cobas Amplicor assay and the ‘in‐house’ PCRs may have two explanations.…”

“…However, the assay was rather disappointing when tested by different investigators with nonrespiratory specimens such as pleural effusions, cerebral spinal fluids and gastric aspirates (29–34), and smear‐negative respiratory samples. The reported sensitivity and specificity values after resolving discordant results (discrepant analysis) ranged from 79.4–91.9% and 85.7–100% for respiratory specimens and 27.3–98.6% and 85.7–100% for nonrespiratory specimens, respectively (1, 35–50).…”