Our study has validated Rv1681 protein as a biomarker of active TB. The detection of full-length Rv1681 protein in the urine of patients with active pulmonary TB by immunoaffinity precipitation supports our initial suggestion that this molecule has promise as a urinary diagnostic biomarker; our detection of the protein in the urine specimens of 11/25 (44%) culture-confirmed TB patients further strengthens this argument. Our study included four types of key clinical negative controls, i.e., healthy individuals, patients with high urinary burdens of E. coli (a frequent colonizer and cause of UTI), patients with non-TB tropical diseases, and patients in whom TB was initially suspected but then ruled out by conventional methods. The clinical specificity of the assay is supported by the complete absence of urine reactivity in the healthy control individuals (most of whom were PPD positive, were from countries in which TB is endemic, and might have had latent TB), patients with E. coli culture-positive urine specimens (particularly given that our polyclonal antibodies were generated against rRv1681 produced in E. coli), and patients with confirmed non-TB tropical diseases. We made the decision to include the TB-ruled-out controls () in the determination of the cutoff because we felt that this was more reflective of real-world application than a lower cutoff calculated using only healthy, E. coli culture-positive, and confirmed non-TB tropical disease controls (). TB-ruled-out controls are a critical population for the evaluation of assay specificity, but gold standard diagnosis in this group is inherently complicated by the limitations of existing diagnostic methods. Our assay detected Rv1681 protein in the urine of 1/21 (4.8%) individuals in this group; further rigorous evaluation of our assay in this type of control population will be key to confirming the specificity of our assay.
It has been proposed that a rapid and widely available diagnostic test for TB with ≥85% sensitivity for smear-positive and smear-negative cases and 97% specificity could save ~400,000 lives annually (15
). While the newly developed Xpert MTB/RIF assay (Cepheid, Inc.) (endorsed by the WHO in 2010 [16
]) offers a large step forward by making sensitive nucleic acid amplification-based diagnosis widely available, it does not satisfy the need for a simple inexpensive POC test (6
); the technology remains prohibitively expensive in some parts of the world, is optimized for sputum specimens, and is not intended for use as a POC device. Urine has the potential to be both a convenient and a high-yield clinical sample for TB diagnosis (18
), given the ease of collection from both adults and children, the fact that collection confers minimal TB transmission risk, and the potential utility for diagnosis of smear-negative and extrapulmonary TB. With these potential advantages in mind, our research has focused on the discovery and evaluation of novel M. tuberculosis
antigens that are produced in vivo
in humans (7
) and mice (19
) during active TB disease, enter the blood circulation, and are ultimately eliminated in the urine. The hypothesis underlying these studies has been that microbial antigens found in vivo
in the bodily fluids of the host would be potentially effective candidate molecules for the development of an antigen detection assay for the diagnosis of active TB.
Precedent for the use of mycobacterial antigens in diagnostic test kits is provided by assays developed to detect a mycobacterial cell wall lipopolysaccharide antigen, lipoarabinomannan (LAM), in urine (21
). Although this precedent supports our hypothesis, LAM tests have shown widely varying sensitivities and specificities (resulting in current utility potentially only for diagnosis of HIV-associated TB in patients with advanced immunodeficiency) (21
), and specificity is potentially hindered by the widespread distribution of LAM molecules across mycobacterial species and potential cross-reaction of anti-LAM antibodies with nonmycobacterial species (21
). In contrast, the specificity of Rv1681 protein for the M. tuberculosis
complex suggests that we can expect high clinical specificity of an assay based on this molecule. In this study, only one of our specimens was from a patient who was confirmed to have a pulmonary infection with non-TB mycobacteria, and it tested negative by ELISA. Further evaluation of our assay among subjects with non-TB mycobacterial disease will be important for confirming assay specificity.
We recognize that a sensitivity of 44% would be insufficient for this ELISA to be clinically useful in its current form. We have anticipated that the concentrations of M. tuberculosis
proteins in urine might be below the achievable limit of detection of conventional ELISA, which is typically in the pg/ml range (24
), and that successful clinical test development will likely require the application of more-sensitive novel protein detection technology; collaborative experiments toward this goal are under way. An additional strategy for increasing clinical sensitivity and specificity would be to combine detection of multiple biomarkers in one assay; in support of this approach, we note that we previously demonstrated the ability to detect the M. tuberculosis
biomarker MT_1694 protein in 37.5% (6/16) of patients with active pulmonary TB using a similar conventional ELISA (8
). Larger clinical studies and a robust fully developed diagnostic assay will be required to validate this biomarker in the field and ultimately to determine its optimal place in TB diagnostic algorithms.
In sum, the M. tuberculosis
Rv1681 protein and the other three proteins that we described previously (7
) are the only M. tuberculosis
protein biomarkers reported to date that have been identified directly from urine specimens from TB patients from regions where TB is endemic and thus are, we feel, strong candidates for development of an antigen detection test for the diagnosis of active TB. Such a test should be able to distinguish between patients with active versus latent disease and would thus provide an important new strategy for control of this global disease. In particular, an antigen detection test that uses urine could potentially allow POC TB diagnosis—not only in adults with pulmonary TB, but also in difficult-to-diagnose populations such as children and adults with extrapulmonary TB. Such a test might also address the critical need for an assay that can monitor response to TB treatment. Our work with Rv1681 protein provides promising evidence that this biomarker, in particular, represents an attractive candidate for the development of a highly sensitive and specific urine antigen detection test.