Validation of Mycobacterium tuberculosis Rv1681 Protein as a Diagnostic Marker of Active Pulmonary Tuberculosis

Abstract

The development of an accurate antigen detection assay for the diagnosis of active tuberculosis (TB) would represent a major clinical advance. Here, we demonstrate that the Mycobacterium tuberculosis Rv1681 protein is a biomarker for active TB with potential diagnostic utility. We initially identified, by mass spectroscopy, peptides from the Rv1681 protein in urine specimens from 4 patients with untreated active TB. Rabbit IgG anti-recombinant Rv1681 detected Rv1681 protein in lysates and culture filtrates of M. tuberculosis and immunoprecipitated it from pooled urine specimens from two TB patients. An enzyme-linked immunosorbent assay formatted with these antibodies detected Rv1681 protein in unconcentrated urine specimens from 11/25 (44%) TB patients and 1/21 (4.8%) subjects in whom TB was initially clinically suspected but then ruled out by conventional methods. Rv1681 protein was not detected in urine specimens from 10 subjects with Escherichia coli-positive urine cultures, 26 subjects with confirmed non-TB tropical diseases (11 with schistosomiasis, 5 with Chagas' disease, and 10 with cutaneous leishmaniasis), and 14 healthy subjects. These results provide strong validation of Rv1681 protein as a promising biomarker for TB diagnosis.T uberculosis (TB) remains the second most common cause of death from an infectious disease in the world. According to a 2012 World Health Organization (WHO) report, there were an estimated 8.7 million new cases of TB globally in 2011 and approximately 1.4 million TB-related deaths (430,000 of which involved HIV-positive individuals) (1). Failure to control the spread of TB is due in large part to failure to adequately detect (and thus to efficiently treat) infectious cases (2); therefore, a major focus of the WHO's global plan to stop TB (3) is the development of new simple and cost-effective diagnostic methods to improve case detection. Historically, culture of Mycobacterium tuberculosis from a clinical specimen has been the gold standard for TB diagnosis; however, it is expensive, time-consuming, and not available worldwide. Thus, in resource-limited settings, diagnosis of active TB relies primarily on clinical evaluation and, where available, microscopic evaluation of sputum specimens. Sputum smears have widely varying sensitivities in different settings (20 to 80%) and are of limited utility in paucibacillary (e.g., pediatric and HIVassociated) TB (3-5). Due to the lack of accessible and effective diagnostic methods, many patients in resource-limited settings are treated empirically based on clinical information alone, exposing some patients to unneeded and potentially toxic drugs and missing others who have active TB.The ideal diagnostic method for tuberculosis would be a lowcost, point-of-care (POC), rapid, simple, and accurate test that could distinguish individuals with active TB from those with latent disease or no infection (including Mycobacterium bovis BCGvaccinated individuals) (2, 6). We previously demonstrated the identification by mass spectroscopy (MS) of four ...

Circulating Mycobacterium bovis Peptides and Host Response Proteins as Biomarkers for Unambiguous Detection of Subclinical Infection

“…During infection, Mycobacterium-specific proteins, peptides, DNA, and lipids are released into the circulation (14,15) and are likely to provide unprecedented specificity to identify M. bovis infection. We hypothesized that a panel of biomarkers, including host and pathogen peptides, would establish a precise test to detect subclinical bTB infection.…”

Identification of Mycobacterial Antigens in Human Urine by Use of Immunoglobulin G Isolated from Sera of Patients with Active Pulmonary Tuberculosis

“…Recently, the Campos-Neto group reported the identification of TB antigens in urine samples, but the biomarker reportedly fell short of its expected performance. with a diagnostic sensitivity of less than 50% (4).…”

“…The presence of a peptide of molybdenum import ATPbinding protein (ModC) was also validated in three out of five independent urine samples by a multiple reaction monitoring (MRM) method. It should be noted that ModC is a protein involved in the metabolism of molybdenum ion, and a molybdenum-associated antigen (MoaA-related protein) was also identified by an independent group (3,4). Figure S3 in the supplemental material shows the tandem mass spectrometry spectrum of a ModC peptide with a molecular ion mass of 464.24 m/z (ϩ2) and a peptide sequence of QLSGGQAQR.…”

“…Because urine is a top-priority biomarker source for point-of-care (POC) testing, systematic approaches for identifying tuberculosis antigens in human urine samples have recently been made (2)(3)(4). Lipoarabinomannan (LAM) is one of the best known biomarkers found in urine, and the assay for detecting LAM in the urine of TB patients is commercially available (5)(6)(7).…”

The identification of tuberculosis biomarkers in human urine samples

“…In another study, KASHINO et al [13] used one-dimensional LC combined with MS/MS to identify four M. tuberculosis-derived proteins in the urine of patients with pulmonary TB. A more recent follow-up urinary proteomics study found that a putative molybdopterin biosynthesis protein (Rv1681) was detected in 11 out of 21 TB patients [14].…”