One of the roadblocks to the eradication of tuberculosis (TB) is the difficulty in identifying patients with latent TB infections. Neither the tuberculin skin test nor interferon-gamma release assays (IGRAs) are capable of distinguishing active from latent infection or predicting the chance of reactivation. Now researchers from the Yonsei University have developed a new multiple-target, real-time reverse transcription-PCR (real-time RT-PCR) TaqMan assay. The whole blood assay targets eight human immune markers and can differentiate active pulmonary TB from latent TB infections.
The World Health Organization reports that one third of the world’s population is latently infected with Mycobacterium tuberculosis (MTB). It has been estimated that in 5% to 10% of latent TB infections individuals, the infection progresses to an active disease state, and the conversion rate is greater in immunosuppressed individuals such as those with HIV. Therefore, rapid diagnostic tests and effective treatment of latent TB infections are important to reduce and control the TB burden.
In previous work, the researchers quantified interferon-? (IFN-?) mRNA expression levels as an indicator of IFN-? levels in an IGRA test. However, the results of IFN-? RT-PCR showed poor specificity and sensitivity, and the test could not be used to distinguish between active and latent TB.
With these results in mind, the team developed a multiple-target RT-PCR TaqMan assay that could target eight human immune markers; Th1-type factors (IFN-?, TNF-?, and IL-2R), Th2-type cytokines (IL-4 and IL-10), and IFN-?-induced chemokines [CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC)]. MTB-specific, antigen-dependent mRNA expression levels were measured in blood samples from 28 patients with active pulmonary TB, 22 with latent TB infections, and 29 non-TB controls.
When five of the human immune markers were evaluated individually, three were found to be suitable for detecting active pulmonary TB, TNF-?, IL-2R, and CXCL10, with sensitivities ranging from 96.43% to 100%. Two individual markers, IL-2R and CXCL10, were able to detect latent TB infections, with sensitivities of 86.36% and 81.82%, respectively.
To optimize sensitivity the team used the assay to simultaneously detect multiple targets. They found that the combination of TNF-?, IL-2R, CSCL9, and CSCL10 could differentiate active pulmonary TB from healthy controls and latent TB infections from healthy controls. More importantly, the combination could differentiate those with active pulmonary TB from those with latent infection.
The team state that the assay has a sensitivity of 100% for active disease and 81.82% for latent TB infections. These results imply that a combination of suitable single markers is very useful for the efficient diagnosis and differentiation of MTB infection.
The researchers underscore that the healthcare threat resulting from the burden of future TB cases emerging from within the latent pool. The current study highlights some of the shortfalls of tuberculin skin test, mainly the risk of false-positive results in patients who have received the bacille Calmette-Guérin vaccine and the need for skilled interpretation of the results in a follow-up visit.
The team cautions that IGRAs are also limited by their potential for false-positive and false-negative results, are technically challenging to perform reproducibly, and have less than ideal sensitivity, especially in those who are immunologically compromised.
The team predict that one day a combination of selected antigen stimulation with multiplexed cytokine, chemokine, and interferon mRNA detection will lead to new ways of evaluating suspected TB patients for current and future disease risk. With one third of the world’s population at risk for recurrence, the investment in such technology is recommended.
Source: Reed Elsevier Group PLC