Findings

Overall statement or summary

I have carefully read the manuscript titled “Discovery of a unique Mycobacterium tuberculosis protein through proteomic analysis of urine from patients with active tuberculosis” and I liked the concept. Diagnosis of a disease is really important as it determines the regime of treatment to be followed.

Strengths

The authors have used proteomic approach to identify a potential biomarker for tuberculosis from the urine of the clinically confirmed cases of tuberculosis. Such studies could lead to potential biomarkers from urine/ serum samples for the deadly disease Tuberculosis. This would be of great practical value as the culture confirmation from the sputum takes time and moreover, it is difficult to get it small children.

Weaknesses

It is essential that a biomarker is always associated with the disease and is not strain specific. To answer this concern, authors have tried to show its prevalence in different populations of Peru, Peru, Vietnam, and South Africa but I am not completely convinced. Nonetheless, the study is designed nicely, with appropriate experimental design and critical discussion including the limitations of the study.

Major points

Following are certain points of concern/ some other limitations of the present study that need to be addressed in my opinion. 1. The antibody-based immunoreactivity of the sera with the peptide to show the prevalence of the peptide antigen is not convincing as antibodies that would be generated inside human would be against a repertoire of epitopes and antibodies are known to show cross-reactivity. It is possible that the antibody reacting with the peptide antigen is raised against a different peptide from the pathogen proteins and is just showing crossreactivity. 2. It would be of interest to know which strain is the causative agent of tuberculosis in the clinically confirmed cases from which the urine samples were collected for proteomic analysis as it could rule out or confirm the strain specificity associated with the presence of peptide antigen in the urine. 3. Authors have identified the peptide from urine and they have even proposed that this could be an attractive candidate for the development of urine antigen assay. It would have been of interest if the authors had confirmed the presence of the peptide in urine samples from other patients also (by any method like that of mass spectroscopy that they have done or by raising antibodies against the recombinant peptide antigen and doing an ELISA using this antibody). They had collected only sera and no urine.

Minor points

1. Authors could have done direct LC-MS of the urine samples to identify the peptide sequence. They could have cleaned up the samples using kits available to get rid of the common proteins found in human urine. The bottom-up approach could have been better.
2. Authors have not commented on how many bands were observed on SDS-PAGE and whether the SDS-PAGE profile was exactly same for the two urine samples and whether or not all the bands were excised and taken for mass spectroscopy. One just guesses that all bands might have been analyzed.
3. Authors could have skipped few self-cited references that talk of biomarkers in Leishmania, they are not that relevant with respect to this study.
4. In materials and methods section, it would have better to use the name of the antigen “TBCG_03312 C-terminus” rather than word antigen.