Tuberculosis Diagnostic Methods: Clinical Applicability, Implementation Challenges, and Integrated Testing Strategies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript provides a comprehensive narrative review of current diagnostic methods for tuberculosis (TB), covering classic microbiological techniques, latent TB diagnostics, lateral flow assays, and a broad range of molecular methods including automated NAATs, LAMP, LPA, and next-generation sequencing approaches. This manuscript would be valuable to clinicians, microbiologists, and public health professionals seeking an overview of TB diagnostic strategies in different resource settings.

The manuscript is presented as a narrative review; however, the added value over existing WHO guidelines and prior comprehensive reviews on TB diagnostics remains insufficiently defined. At present, the article mostly summarizes established information (test principles, performance characteristics, and WHO-recommended use) that is already available in current guidelines and existing review papers, and it is not clear what novel perspective, framework, or synthesis this review contributes. I encourage the authors to explicitly state what specific gap in the literature they aim to address and to highlight throughout the text how their analysis goes beyond simply reiterating existing WHO recommendations.

The NGS section is valuable but could further distinguish clearly between current routine implementation and future potential.

Authors should add a brief table listing core guidelines and landmark studies by diagnostic category. For each category, authors should add the key limitations (eg: performance in HIV, children, extrapulmonary TB; operational constraints; biosafety needs etc) and evidence gaps. Consider including a summary table that aligns test type, sample type, turnaround time, required infrastructure, approximate cost tier (low/medium/high), and primary clinical use (screening, initial diagnosis, drug-resistance testing, surveillance). Then this would greatly enhance the practical usability of the review for clinicians and laboratory managers.

Author Response

Reply to reviewer 1 

We thank the reviewer for the valuable comments. Below, we provide responses to each point raised. We hope these address the reviewer’s concerns. We are willing to make further revisions if necessary.

Point 1- I encourage the authors to explicitly state what specific gap in the literature they aim to address and to highlight throughout the text how their analysis goes beyond simply reiterating existing WHO recommendations.

Answer 1 - We thank the reviewer for encouraging us to improve the text on this point. We believe that the manuscript has been strengthened by incorporating the issues raised during the review. We have clarified this point more explicitly, particularly in the final paragraph of the “Introduction” and at the end of the “Practical implications for the clinical laboratory”, which are highlighted in gray.

Point 2 -The NGS section is valuable but could further distinguish clearly between current routine implementation and future potential.

Answer 2 - We agree with the reviewer and have therefore supplemented the NGS section with two additional paragraphs (the 8th and 9th), highlighted in gray, addressing both the current routine application and future perspectives of NGS. For the reviewer’s convenience, we reproduce the revised text below:

At present, routine implementation is mainly restricted to reference laboratories and national surveillance programs, where tNGS is used to complement or replace culture-based drug susceptibility testing, particularly in cases with suspected drug resistance or inconclusive molecular results [58,105,106]. In contrast, mNGS currently represents a technology with important future potential rather than routine clinical use. mNGS enables hypothesis-free pathogen detection directly from clinical specimens and may be especially useful for paucibacillary or extrapulmonary TB and for the investigation of differential diagnoses or co-infections [107-109]. As a result, mNGS is presently applied mostly in research contexts or selected complex clinical cases, while further technical optimization, cost reduction, and validation studies will be required before widespread adoption in TB diagnostic workflows can be achieved [108,109].

Although NGS offers expanded coverage of resistance-associated mutations and the potential for comprehensive genomic characterization, its clinical implementation is still constrained by important limitations. Routine use requires specialized bioinformatics pipelines, trained personnel, and high-complexity laboratory infrastructure, which restricts availability in resource-limited settings [105-106]. In addition, the risk of cross-sample or environmental contamination during nucleic acid extraction and library preparation may compromise result reliability, particularly in laboratories without strict quality assurance procedures [106,107]. 

Point 3 - Authors should add a brief table listing core guidelines and landmark studies by diagnostic category. For each category, authors should add the key limitations (eg: performance in HIV, children, extrapulmonary TB; operational constraints; biosafety needs etc) and evidence gaps. 

Answer 3 - In response to this suggestion, a table has been added to the “Practical implications for the clinical laboratory” section, including the requested data. The table is highlighted in gray.

Point 4 - Consider including a summary table that aligns test type, sample type, turnaround time, required infrastructure, approximate cost tier (low/medium/high), and primary clinical use (screening, initial diagnosis, drug-resistance testing, surveillance). Then this would greatly enhance the practical usability of the review for clinicians and laboratory managers.

Answer 4 - We hope that Table 1, added in response to Point 3, adequately addresses this comment.

We would like to thank the reviewer once again for their valuable comments. In addition to revising the main text of the review, we considered it necessary to adjust the manuscript’s title and abstract. Accordingly, these sections were also revised to ensure greater coherence and homogeneity throughout the manuscript. These sections also are highlighted in gray.

Reviewer 2 Report

Comments and Suggestions for Authors

In this review article, authors state that TB is a major global health threat, worsened by HIV and drug-resistant strains. This review outlines various laboratory diagnostic methods for active and latent TB, evaluating their clinical application, performance, and integration into healthcare settings. It discusses traditional methods, like smear microscopy and culture, alongside modern techniques such as nucleic acid amplification tests (NAATs), loop-mediated isothermal amplification (LAMP), line probe assays (LPA), next-generation sequencing (NGS), and lateral flow assays. Each method’s strengths and limitations are assessed based on infrastructure, resources, and epidemiological context. While traditional methods are useful in certain situations, molecular technologies offer greater sensitivity and faster results. The article highlights the need for complementary diagnostic strategies within hybrid testing algorithms to enhance resource use, ensure accuracy, promote access, and facilitate early treatment, ultimately aiding TB control efforts.

Here are some of my comments:

1. The review examines diagnostic approaches, excluding considerations of cost, feasibility, or necessary infrastructure, particularly regarding NAAT platforms such as Xpert, Truenat, and LPA, as well as NGS and tNGS systems. Given that the greatest burden of TB is observed in resource-constrained settings, the exclusion of economic and practical implementation factors substantially limits the practical relevance of the conclusions.
2. Sequencing technologies are presented as generally applicable; however, critical limitations, including the complexity of bioinformatics, the risk of contamination, the requirements for infrastructure and staffing, reduced yield in cases of paucibacillary disease, and difficulties in data interpretation, are not adequately addressed.
3. Furthermore, the limited discussion of extrapulmonary and pediatric TB, despite their unique diagnostic challenges such as atypical specimens, paucibacillary disease, and decreased test sensitivity, necessitates dedicated analysis.
4. The discussion of latent TB is presented separately from active TB diagnostic methods, thereby neglecting crucial aspects such as screening protocols, preventative initiatives, the assessment of progression risk, and considerations specific to pediatric populations, which diminishes both conceptual clarity and clinical applicability.
5. Novel diagnostic methodologies, including host-response biomarkers, proteomics/metabolomics, AI-assisted imaging, and nano diagnostics, are not addressed, resulting in a review that is predominantly retrospective in nature, rather than forward-looking.
6. The proposed workflows are characterized by oversimplification and lack practical utility, as they fail to account for variations in resource availability, patient stratification, treatments beyond pulmonary involvement, pediatric care, or specific challenges associated with HIV.
7. The WHO's guidelines are presented without a critical appraisal of the underlying evidence, encompassing its strengths and limitations, regional disparities, or existing areas of contention.
8. Moreover, the review does not sufficiently address issues such as false positives, false negatives, and contradictory findings.
9. The review also lacks important methodological details. These include a clear search strategy, the criteria used to include or exclude studies, a way to assess the quality of the evidence, and an evaluation of potential biases. As a result, the transparency, reproducibility, and scientific reliability of the findings are reduced.

 

Minor comments:
1. Some assertions are insufficiently supported by strong or updated citations.

2. Provide clearer labeling in Figure for improved visual clarity.
3. Terms such as “rapid test” and “reliable” should be more precisely defined using quantitative or standard diagnostic metrics.
4. The issues of false positives/negatives are mentioned but not discussed in sufficient detail.
5. A table could be inserted for clearer side by side comparison of diagnostic methods would enhance interpretive value.

Author Response

Reply to reviewer 2

We thank the reviewer for the valuable comments. Below, we provide responses to each point raised. We hope these address the reviewer’s concerns. We are willing to make further revisions if necessary.

Point 1 - The review examines diagnostic approaches, excluding considerations of cost, feasibility, or necessary infrastructure, particularly regarding NAAT platforms such as Xpert, Truenat, and LPA, as well as NGS and tNGS systems. Given that the greatest burden of TB is observed in resource-constrained settings, the exclusion of economic and practical implementation factors substantially limits the practical relevance of the conclusions.

Answer 1 - We agree with the reviewer and have addressed these points by adding a new table highlighted in gray in manuscript (Table 1). This table includes a five-level cost classification (from very low to very high), a summary of key limitations for each diagnostic approach, and the required biosafety level, thereby indicating the laboratory infrastructure needed for implementation.

Point 2 - Sequencing technologies are presented as generally applicable; however, critical limitations, including the complexity of bioinformatics, the risk of contamination, the requirements for infrastructure and staffing, reduced yield in cases of paucibacillary disease, and difficulties in data interpretation, are not adequately addressed.

Answer 2 - We agree with the reviewer that sequencing technologies, particularly NGS, require a more detailed discussion of their limitations. We have therefore expanded the “Next-generation sequencing (NGS)” section by adding paragraphs 9 and 10, highlighted in gray for ease of reference. These additions address NGS performance in paucibacillary samples, the need for specialized training of laboratory personnel, and key aspects of bioinformatics workflows and data interpretation, among other points.

Point 3 - Furthermore, the limited discussion of extrapulmonary and pediatric TB, despite their unique diagnostic challenges such as atypical specimens, paucibacillary disease, and decreased test sensitivity, necessitates dedicated analysis.

Answer 3 – In response to this suggestion, we added a fifth paragraph to Section “Automated nucleic acid amplification tests (NAAT)”, addressing samples with low bacillary burden. In addition, the twelfth and thirteenth paragraphs were added to the section “Practical implications for the clinical laboratory”, discussing extrapulmonary and pediatric tuberculosis. All three paragraphs are highlighted in gray for ease of reference.

Point 4 - The discussion of latent TB is presented separately from active TB diagnostic methods, thereby neglecting crucial aspects such as screening protocols, preventative initiatives, the assessment of progression risk, and considerations specific to pediatric populations, which diminishes both conceptual clarity and clinical applicability.

Answer 4 -  We agree with the reviewer that the discussion on latent tuberculosis should be expanded to address the clinical applicability of diagnostic methods in this context. Accordingly, we have added two paragraphs (paragraphs 8 and 9) to the section “Practical implications for the clinical laboratory”, discussing the applicability of selected tests for latent TB. These additions are highlighted in gray for clarity.

Point 5 - Novel diagnostic methodologies, including host-response biomarkers, proteomics/metabolomics, AI-assisted imaging, and nano diagnostics, are not addressed, resulting in a review that is predominantly retrospective in nature, rather than forward-looking.

Answer 5 – We agree with the reviewer that emerging diagnostic approaches were not sufficiently addressed in the initial version, which may have limited the forward-looking perspective of the review. To address this important point, we have added a new subsection entitled “Emerging diagnostic methodologies”. This new section is highlighted in gray.

Point 6 - The proposed workflows are characterized by oversimplification and lack practical utility, as they fail to account for variations in resource availability, patient stratification, treatments beyond pulmonary involvement, pediatric care, or specific challenges associated with HIV.

Answer 6 – We acknowledge the reviewer’s concern regarding the limited practical utility of the proposed workflow. Given its repetitive nature and oversimplification, the flowchart has been removed. The information previously presented in the flowchart has been incorporated into Table 1, which now provides a more informative and practical overview, including resource requirements for each diagnostic test.

Point 7 - The WHO's guidelines are presented without a critical appraisal of the underlying evidence, encompassing its strengths and limitations, regional disparities, or existing areas of contention.

Answer 7 – We acknowledge the reviewer’s concern regarding the presentation of the WHO guidelines without a critical assessment of the underlying evidence. In response, we have added two paragraphs (paragraphs 14 and 15) to the section “Practical implications for the clinical laboratory”, providing a more critical discussion that addresses omissions in the WHO text, including the use of metagenomic approaches and considerations related to latent TB. These additions are highlighted in gray for clarity.

In addition, we note that occupational TB is not explicitly addressed in the WHO guidelines. This topic is discussed in paragraph 11 of the same section, which is also highlighted in gray.

Point 8 - Moreover, the review does not sufficiently address issues such as false positives, false negatives, and contradictory findings.

Answer 8 – In response to this comment, we have added the following sentence to paragraph 11 of the section “Practical implications for the clinical laboratory”: “Another important consideration is the heterogeneity in test performance and the occurrence of false-positive and false-negative results. No single method is sufficient for all clinical scenarios; therefore, integrated diagnostic workflows are essential to mitigate individual test limitations and support context-specific decision-making.” This addition is highlighted in gray for clarity. 

In addition, the final paragraph of each diagnostic methods section now explicitly addresses issues related to false-positive and false-negative results, as well as contradictory findings, for each methodology individually. These additions are highlighted in green.

Point 9 - The review also lacks important methodological details. These include a clear search strategy, the criteria used to include or exclude studies, a way to assess the quality of the evidence, and an evaluation of potential biases. As a result, the transparency, reproducibility, and scientific reliability of the findings are reduced.

Answer 9 – In response to this comment, we have substantially revised the “Search Strategy” section to improve transparency and reproducibility by clearly detailing the search strategy, inclusion and exclusion criteria, assessment of evidence quality, and potential sources of bias. This section is highlighted in gray.

We would like to thank the reviewer once again for their valuable comments. In addition to revising the main text of the review, we considered it necessary to adjust the manuscript’s title and abstract. Accordingly, these sections were also revised to ensure greater coherence and homogeneity throughout the manuscript. These sections also are highlighted in gray for clarity.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript has significantly improved after addressing the major comments. I don't have any more comments.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all the comments. The revisions are clear and scientifically sound and have improved the clarity and quality of the manuscript. The responses are well justified, and no further concerns remain. I recommend acceptance of the manuscript for publication.