Detection and significance of anti-Mycobacterium tuberculosis specific IgG antibody response for the diagnosis of pulmonary tuberculosis using enzyme-linked immunosorbent assay
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Objective: Evaluation of an ELISA test for detection of IgG antibody response using in-house prepared Mycobacterium tuberculosis H37Rv soluble extract (MTSE) as antigen for rapid diagnosis of pulmonary tuberculosis and its clinical usefulness. Methods: In this study, a total of 758 pulmonary tuberculosis (TB) patients (652 AFB-positive and 106 AFB-negative), 276 healthy controls, and 43 pulmonary infectious disease controls other than TB were recruited. IgG antibody level against MTB soluble extract was measured in sera samples of all study groups using an ELISA test. The level of IgG antibody responses was compared among groups by the Kruskal-Wallis test. The pairwise comparison was made by the Mann-Whitney test, A positive score was represented by optical density above the cut–off value which was calculated from OD values of healthy controls by adding 2SD to the mean OD value. The evaluation of diagnostic value was considered based on sensitivity and specificity. Results: Significantly higher levels of IgG antibody response were observed in PTB patients compared to healthy control and non-TB other pulmonary infectious disease control groups (p value<0.0001). The percent positivity for the IgG antibody response was higher in AFB-positive 574/652 (88.04%) and 79/106 (74.53%) AFB-negative PTB patients as compared to healthy control 9/276 (3.26%) and non-TB other pulmonary infectious disease control 3/43 (6.97%). The sensitivity of the test in PTB patients (AFB-positive and AFB-negative) was 86.15% (95% CI; 83.48-88.53), and the specificity was 96.74% (95% CI; 93.90-98.50). Conclusion: This developed immunological test could be an efficient test in detecting IgG antibody response in PTB patients. Further, this test could be useful for diagnosing AFB-negative presumptive TB cases.
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We have now received expert reviewer comments for your paper, and based on these comments and my assessment, I am sorry to inform you that your manuscript is not suitable for publication in Access Microbiology. The reviewers raise concerns regarding the scientific rigour and experimental design of the work.
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Comments to Author
Even though the article has been improved by the authors this work is still lacking in certain areas highlighted in previous reviews: 1. Timelines and which patient groups were assessed when could have been presented in a format that is clearer 2. Without comparisons to a commonly used control one cannot truly test if this proposed method is more specific/sensitive 3. For 90% power the authors claim that 283 participants were required per group but there were far less participants in the PTB AFB-negative and the Non-TB Other pulmonary group.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Comments to Author
Even though the article has been improved by the authors this work is still lacking in certain areas highlighted in previous reviews: 1. Timelines and which patient groups were assessed when could have been presented in a format that is clearer 2. Without comparisons to a commonly used control one cannot truly test if this proposed method is more specific/sensitive 3. For 90% power the authors claim that 283 participants were required per group but there were far less participants in the PTB AFB-negative and the Non-TB Other pulmonary group.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
Yes: There are several methodological issues that have been highlighted in the reviews including lack of proper controls, not testing the controls in the same manner as the "test" and lack of pre-research power calculations.
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
The authors have carried out some of the recommended changes including title and abstract changes, revision of some of the grammatical errors, inclusion of additional healthy controls, amendments of Figure titles and inclusion of some of the confidence interval values. Nonetheless this work still has several pending issues including major methodological shortcomings: 1. The flow of the article is at times still lacking making it difficult to follow with several disjointed sentences. 2. There are still quite a few sentences that could have been written in a more academically appropriate manner eg. "The tuberculin skin test (TST) is of limited utility in the diagnosis of active TB 76 due to both false negative and false positive results [13]" I would have referred to the reported specificity and …
Comments to Author
The authors have carried out some of the recommended changes including title and abstract changes, revision of some of the grammatical errors, inclusion of additional healthy controls, amendments of Figure titles and inclusion of some of the confidence interval values. Nonetheless this work still has several pending issues including major methodological shortcomings: 1. The flow of the article is at times still lacking making it difficult to follow with several disjointed sentences. 2. There are still quite a few sentences that could have been written in a more academically appropriate manner eg. "The tuberculin skin test (TST) is of limited utility in the diagnosis of active TB 76 due to both false negative and false positive results [13]" I would have referred to the reported specificity and sensitivity of this test. I am sure there is systematic review with meta-analysis that has a reliable value that you can quote 3. Additional healthy controls. The timings of the different rounds of recruitment need to be specified in the methods to be transparent that further subjects were recruited. As this also leads to additional bi issues as you've only recruited additional "healthy" controls so it was very clear what results you were expecting in this group even if possibly in the initial group there was some form of very partial bias according to your statement in the review document. These issues should have been looked at the study design stage. 4. I still believe that it is a major methodological flaw that the traditional test was not performed on all the subjects. You cannot make claims that the specificity and sensitivity are better with your proposed method without effectively comparing it to the standard method used. 5. Even though the confidence intervals of some of the values have been included, the values for the most important numerical values in the paper ie Figure 1 6. The authors claim that a power of 90% was calculated but no information of what method/tools have been used to assess this
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
Yes: I am mainly concerned re the methods used and the amount of bias in this and the lack of proper comparison to the method that they'd aimed to improve on.
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
It is successfully revised to my questions.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
This research looks into assessing the use of a novel antibody to aid TB diagnosis. The findings are important in tackling current challenges. None the less the paper requires several clarifications and amendments: 1. Title and Abstract I would include information on what sort of antibody or antibodies and/or respective antigen are being detected as currently this is not clear in the title and abstract 2. Introduction: A. See comments on repetition, sentences and grammar in point 7 Eg. "The diagnosis of pulmonary tuberculosis (PTB) is primarily based on the detection of MTB in sputum smears (acid-fast staining) and bacilli culture. Microscopy is largely available but missing sensitivity and specificity remains a major problem in several samples and MTB culture 69 takes as much as 6-8 weeks for …
Comments to Author
This research looks into assessing the use of a novel antibody to aid TB diagnosis. The findings are important in tackling current challenges. None the less the paper requires several clarifications and amendments: 1. Title and Abstract I would include information on what sort of antibody or antibodies and/or respective antigen are being detected as currently this is not clear in the title and abstract 2. Introduction: A. See comments on repetition, sentences and grammar in point 7 Eg. "The diagnosis of pulmonary tuberculosis (PTB) is primarily based on the detection of MTB in sputum smears (acid-fast staining) and bacilli culture. Microscopy is largely available but missing sensitivity and specificity remains a major problem in several samples and MTB culture 69 takes as much as 6-8 weeks for results. This delay makes the patient more infectious and more progressing in the disease [12]." "Interferon gamma release assay (IGRA) is unable to satisfactorily differentiate latent TB from active TB [14] and Nucleic Acid Amplification Test (NAAT) is a cost-effective method for TB diagnosis by increasing the case finding in substantially high-burden countries [15]." Require rewording. B. Information in "61 MTB is unique among bacterial pathogens in that it displays a wide array of complex lipids and lipoglycan on its cell surface [10]. These exclusive cell wall lipids are known to play an important role in pathogenesis. MTB has many virulent factors that increase the severity of the disease [11]. " Can be included into previous paragraph to decrease repetition. Retain the last sentence "Therefore, accurate and rapid diagnosis is of great importance for early treatment and containment of this disease." to retain flow to the next paragraph on detection. 3. Methodological rigour, reproducibility and availability of underlying data A. "Clinical diagnosis, AFB smear positivity was checked in two specimens of sputum (one of them 95 was a morning sputum specimen and another one was collected on the spot) by Ziehl-Neelsen 96 (ZN) staining." What do you mean on by on the spot? Does this mean that timings vary for the second reading? Please specify timings B. "All the healthy volunteers were physically checked by clinicians as healthy" What did this check include? Were they subjected to an AFB smear? Clarify what tests were done to confirm that the volunteers were healthy/non-TB patients C. Table 1 title adjust to "Clinical and demographic information of healthy controls (HC) and active pulmonary tuberculosis patients (PTB)." D. Table 1: High Risk Behaviour: Alcoholic : What definition of alcoholic did you use? Generally alcohol consumption is classified as units per week in scientific literature. Kindly amend to units consumed. Smoking: What sort of smoking? How much? Generally this category is divided into non-smoker, smoker, ex-smoker and the smoker and ex-smokers further divided into amount and type of smoking. Tobacco: Is this chewing tobaccos? Please specify E. Specify type of vortexing and for how long and when F. Were all samples stored for the same amount of time? Why -20 and not -40? G. How was H37Rv produced and validated? Or was it bought? If so please specify brand supplier etc H. "The experiment was 175 replicated three times." Were replications carried as 3 independent experiments? I: Was any blinding introduced when testing the samples and during analysis? 4. Presentation of results A. Why wasn't the specificity and sensitivity of this antibody not compared to other commercially available tests on the market using the same samples? B. Has the power been calculated for the results? If so how much is it? C. Include 95% confidence interval in data tables 5. How the style and organization of the paper communicates and represents key findings A. Mean age between the healthy and the PTB patient are significantly different. It would be best to recruit a few more healthy controls to get comparable ages after discussion with a statistician. B. I would have a general discussion with a statistician to see if there are any co founding variables which are effecting your results 6. Literature analysis or discussion A. Kindly add more information about sensitivity and specifity of currently used methods of detection B. Had this antibody been tested by others? If so what were the findings? C. Highlight limitations of this study and how this effects further research and your findings 7. Other General comments: A. Several repetition and lengthy sentences are present throughout the document. Kindly also review the article's grammar. Eg. "The MTB enters the host by the respiratory tract and once reaches the lungs, is phagocytosed by macrophages"
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
This manuscript describes the diagnostic potential through the detection of antibodies against the Mth sonic extract antigen in sera from AFB-confirmed active TB (n=652) and healthy controls (n=251) by ELISA. They have obtained 88.5% sensitivity and 97.61% specificity. Serodiagnosis for TB has been applied long time because of its easy and simple method. However, successful results and kits could not be obtained and developed. I think that the only merit of this manuscript is enough sample numbers. However, too extreme a research group was chosen, which resulted in deviation results. Healthy control should be grouped in tuberculin-negative and positive groups, and a group of AFB-negative TB patients must be included, and also other pulmonary infectious diseases are also included as control group …
Comments to Author
This manuscript describes the diagnostic potential through the detection of antibodies against the Mth sonic extract antigen in sera from AFB-confirmed active TB (n=652) and healthy controls (n=251) by ELISA. They have obtained 88.5% sensitivity and 97.61% specificity. Serodiagnosis for TB has been applied long time because of its easy and simple method. However, successful results and kits could not be obtained and developed. I think that the only merit of this manuscript is enough sample numbers. However, too extreme a research group was chosen, which resulted in deviation results. Healthy control should be grouped in tuberculin-negative and positive groups, and a group of AFB-negative TB patients must be included, and also other pulmonary infectious diseases are also included as control group because the antigen used for antibody detection is Mtb total extracts, which contain complex antigens cross-reacting with other pathogens. In fact, we can find many papers similar to this manuscript, so new findings or designs are needed. The minor comment is that it is a very important issue when the serum samples are taken; before and after treatment. Usually, antibody titers are increased by treatment and higher in chronic conditions than early early-diagnosis patients. By the published papers and my experiments, early diagnostic patients have shown very low antibody titers, but these patients should be practical major targets for serodiagnosis in clinical fields rather than chronic TB patients. That's why this issue makes serodiagnosis difficult.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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