Cost-effective identification of Mycobacterium tuberculosis complex using microscopic morphology and rapid tests.

BACKGROUND: The rapid diagnosis of tuberculosis (TB) by detecting and identifying Mycobacterium tuberculosis complex (MTC) in clinical culture isolates can be achieved by a combination of rapid tests. OBJECTIVE: To propose a cost-effective laboratory protocol for MTC identification. DESIGN: MTC (n = 278) was identified using microscopic morphology, two immunochromatographic assays (ICAs) (Tibilia™ and MeDiPro(®) M. tuberculosis Antigen Rapid Test) and the strand displacement amplification (SDA) method (ProbeTec), and the results were compared. RESULTS: Microscopic morphology (cord-like) had a sensitivity of 99.3%, a specificity of 84.3%, a positive predictive value (PPV) of 88.2% and a negative predictive value (NPV) of 99.1%. The overall sensitivity/specificity of the Tibilia, MeDiPro and ProbeTec assays were respectively 98.7%/98.4%, 88.0%/85.2% and 97.4%/98.4%. The PPV/NPV for Tibilia, MeDiPro and ProbeTec were respectively 98.7%/98.4%, 87.4%/85.8% and 98.7%/96.8%. Cord-like microscopy was the least expensive method and could be used for the identification of MTC. ICA offers cost-effective screening compared to the SDA method. Tibilia performed better than MeDiPro, while its diagnostic value was similar to the SDA method. CONCLUSION: We recommend a combination of microscopic morphology and Tibilia to further improve the sensitivity and PPV of MTC identification at lower cost.

Combination of molecular assay and clinical evaluation for early confirmation of tuberculosis cases.

The cost-effectiveness of the ProbeTec ET Direct TB assay (DTB) was compared with that of culture for detection of Mycobacterium tuberculosis complex in 361 acid-fast stain-positive respiratory specimens. The overall sensitivity, specificity, positive predictive value and negative predictive value of DTB were 97.7%, 86.6%, 87.2% and 97.6%, respectively. When clinical evaluation was added to DTB, the specificity and positive predictive value of DTB increased to 94.7% and 95.4%, respectively. Treatment costs of $133,521 would have been saved in this cohort if DTB, instead of culture results, had been used to eliminate 'false-positive' smear results.

Detection of M. tuberculosis using DNA chips combined with an image analysis system.

OBJECTIVE: To develop a packaged DNA chip assay (the DR. MTBC Screen assay) for direct detection of the Mycobacterium tuberculosis complex. DESIGN: We described a DNA chip assay based on the IS6110 gene that can be used for the detection of M. tuberculosis complex. Probes were spotted onto the polystyrene strips in the wells of 96-well microtitre plates and used for hybridisation with biotin-labelled amplicon to yield a pattern of visualised positive spots. The plate image was scanned, analysed and interpreted automatically. RESULTS: The results corresponded well with those obtained by conventional culture as well as clinical diagnosis, with sensitivity and specificity rates of respectively 83.8% and 94.2%, and 84.6% and 96.3%. CONCLUSION: We conclude that the DR. MTBC Screen assay can detect M. tuberculosis complex rapidly in respiratory specimens, readily adapts to routine work and provides a flexible choice to meet different cost-effectiveness and automation needs in TB-endemic countries. The cost for reagents is around US$10 per sample.

Dideoxy fingerprinting for rapid screening of rpoB gene mutations in clinical isolates of Mycobacterium tuberculosis.

Rifampin is a key component of therapeutic regimens for tuberculosis control, and a marker for multidrug resistance of Mycobacterium tuberculosis. Mutations responsible for conferring rifampin resistance in M. tuberculosis are known to occur in a 69-bp region of the rpoB gene. In this study, we assessed the accuracy of dideoxy fingerprinting (ddF), a hybrid technique employing elements of dideoxy sequencing and single-strand polymorphism analysis, for rapid screening of rifampin resistance in clinical isolates of M. tuberculosis. This technique was used to analyze 72 M. tuberculosis isolates. The results were compared with those of automated dideoxy sequencing and the antibiotic resistance profile (determined with the BACTEC system). Of the 72 isolates, 50 were rifampin resistant. The ddF findings were completely consistent with those of dideoxy sequencing in all isolates. In 68 (94%) isolates, the ddF findings were consistent with the rifampin resistance status determined with the BACTEC system; all four isolates with inconsistent results had no mutation in the 69-bp region, but were resistant to rifampin. Our findings suggest that ddF accurately detects mutations in the rifampin resistance-associated 69-bp region of the rpoB gene in clinical isolates of M. tuberculosis, and may be a valuable screening tool for rifampin resistance.

Evaluation of polymerase chain reaction-restriction enzyme analysis of mycobacteria cultured in BACTEC 12B bottles.

Polymerase chain reaction with restriction enzyme analysis (PRA) was first tested on 15 reference strains and 50 subcultured clinical isolates of mycobacteria according to the reference algorithm by Telenti et al [1]. Next, we evaluated the application of this method to 108 isolates from liquid media (BACTEC 12B). Of them, 15 M. tuberculosis complex and 81 mycobacteria other than tuberculosis (MOTT) had comparable results with both PRA and the BACTEC 460 TB systems. However, seven M. tuberculosis complex and three potentially pathogenic MOTT were identified by PRA rather than the BACTEC TB system. PRA seems to be an efficient method for the identification of mycobacteria to the species level and a good aid to detect potentially pathogenic mycobacteria, especially in mixed mycobacterial cultures.