<xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Genotypic study of isolated resistance to isoniazid in the Mycobacterium tuberculosis&#160; complex in&#160; a Moroccan hospital </xhtml:span>

  1. Amine Amri
  2. Elmostafa BENAISSA
  3. Yassine BENLAHLOU
  4. Fatna BSAIBIS
  5. Adil MALEB
  6. Mariama CHADLI
  7. Mostafa ELOUENASS

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Abstract

Introduction: Despite the introduction 40 years ago of effective and low-cost treatment for tuberculosis, morbidity and mortality from this disease remain substantial worldwide. According to the WHO, tuberculosis is once again the leading cause of death worldwide from a single infectious agent. In 2023, TB caused approximately 1.25 million deaths. In Morocco, the number of new tuberculosis cases rose from 30,897 in 2017 to 35,000 in 2019.The incidence of multidrug-resistant or rifampicin-resistant tuberculosis was estimated at 1.7 per 100,000 inhabitants, Isoniazid is a cornerstone of first-line TB treatment, and resistance to it even in the absence of rifampicin resistance is associated with delayed treatment response, higher rates of treatment failure or relapse,and increased risk of progression to MDR-TB if not promptly identified and appropriately managed. Moreover, current diagnostic algorithms in many settings, including Morocco, may miss INH monoresistance due to their reliance on rapid molecular tests that primarily detect rifampicin resistance. further emphasizing the emerging threat of drug-resistant TB. Despite this, national data on isoniazid monoresistance remain scarce. Given the increasing burden of TB and the critical importance of early detection of drug resistance, it is essential to better understand patterns of resistance beyond rifampicin. It is within this context that we conducted the present study, which aims to investigate isoniazid resistance in tuberculosis cases over a period of three years.     Materials and Methods This is a retrospective study conducted at the Bacteriology Department of Mohammed V Military Instruction Hospital over a period of 3 years. Data were collected via the laboratory information system. Clinical samples underwent treatment using both conventional bacteriological methods and molecular techniques. The study of resistance to major anti-tuberculosis drugs was performed using the reverse hybridization technique, specifically the HAIN method (GenoType® MTBDRplus by Hain Lifescience. Statistical analysis was performed using IBM SPSS Statistics19 and Microsoft Excel 2019                                       Results: The study involved 464 patients treated for pulmonary and extrapulmonary tuberculosis, including both new cases and those previously treated with positive cultures. The mean age of the patients was 42.2 years, with a range from 8 to 88 years. There was a predominance of males at 74%, with a sex ratio of 2.8.                                                                                    Pulmonary sputum samples accounted for 84.8% of the cases, where as extrapulmonary samples representedonly15.2%,the positivity rates for direct examination and culture across all samples were74%and100%, respectively. Isoniazid resistance had a prevalence of 9% (43/464). Genetic mutations observed indicated that 63% of the clinical isolates resistant to INH had mutations in the katG gene, while 37% had mutations in the inhA gene. Conclusion The increasing prevalence of Mycobacterium tuberculosis complex strains resistant to one or more first-line anti-tuberculosis drugs highlights the urgent need for targeted and ongoing epidemiological surveillance. In this study, we found that isoniazid resistance affected 9% of tuberculosis cases over the three-year period, underscoring a significant yet under recognized threat to TB control efforts in Morocco. Molecular analysis revealed that the majority of resistant strains carried mutations in the katG gene, with a smaller proportion exhibiting mutations in the inhA promoter region.

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  1. Version published to 10.1099/acmi.0.000928.v3 on Access Microbiology
  2. Access Microbiology

    Dear authors, thank you for submitting your revised manuscript to Access Microbiology. Unfortunately, the previous reviewers were unable to review your revised manuscript leading to a small delay in assigning a new reviewer. It was noted that there could be a stronger alignment to the study’s aims throughout the manuscript, especially in the conclusions section. Please find the reviewer’s comments at the end of this email, for your attention. In addition, please ensure that figure legends are expanded further so that they are self-explanatory and independent of the text (i.e. a reader can understand each figure without needing to refer to the text). The figure changes were appreciated, but I suggest reverting them back to the version 1 figures, as the borders are not required. Furthermore, please change figure 2 to a two-dimensional chart for consistency and ensure the background of figure 3 is white/plain and not grey, this will improve their presentation. The conclusion section contains discussion-like text. I suggest reviewing your conclusions section so that it is more concise and discussion topics moved into the discussion section.

  3. Access Microbiology

    Comments to Author

    General Overview This manuscript addresses an important topic and provides valuable data on isoniazid resistance in Mycobacterium tuberculosis in Morocco. The focus on mutation patterns offers scientific value and relevance to the field. However, a more transparent structure, a stronger alignment with the study's aim, and more specific reporting and interpretation of the findings would significantly improve the study's overall impact and clarity. ABSTRACT The parenthetical reference to 30,897 cases in 2017 appears to suggest a comparison with the 2019 figure 35,000 cases, but the comparison is not clearly stated. Consider rephrasing to explicitly highlight the increase and its relevance to the national TB burden. Lines 16-17 Consider adding a bridging sentence for a smoother transition and better connect the rising burden of TB and drug resistance with the study's focus. Lines 16-21 The study of isoniazid monoresistance still requires a clearer justification. While I understand that national data on this form of resistance is limited, the manuscript does not sufficiently explain why isoniazid monoresistance deserves attention. Why is it important to study this form of resistance over a 3-year period? Clarifying the intended impact or purpose of the study would strengthen its overall relevance and scientific contribution. Lines 19-21 The conclusion does not specifically reflect the main findings of the study, which focused on isoniazid monoresistance. It refers broadly to "strains resistant to one or more first-line drugs,". Consider rephrasing the conclusion to emphasize the reported 9% prevalence of isoniazid resistance and the genetic mutations identified (katG and inhA), as well as their relevance for TB control and surveillance. Lines 41-44 INTRODUCTION The Introduction presents useful information on molecular diagnostic tools such as GeneXpert and MTBDRplus. However, the connection between this background and the study's focus on isoniazid resistance could be more clearly established. Consider strengthening the link between the background and the study aim to make the rationale more compelling. To enhance the clarity and relevance of the aim, I also recommend elaborating on the potential clinical, diagnostic, or public health implications of isoniazid monoresistance. According to the WHO, tuberculosis is once again the leading cause of death worldwide from a single infectious agent. In 2023, TB caused approximately 1.25 million deaths, surpassing COVID-19. Check the WHO report: https://www.who.int/news-room/fact-sheets/detail/tuberculosis?utm_source=chatgpt.com. In general, several pieces of data presented in this section lack proper citation, compromising the manuscript's credibility. Appropriate and verifiable references should support all factual statements. Materials and Methods It might be a good idea to split the "Materials and Methods" section into appropriate subsections, such as Study Design and Setting, Laboratory Methods, and Data Analysis. This would help distinguish between the design aspects of the study and the actual methods used, thereby improving clarity. The section provides a helpful general overview of the GenoType MTBDRplus test (Lines 107-124). However, it currently reads more like background information than a detailed description of how the method was applied in this specific study. To enhance clarity and replicability, I suggest focusing more on study-specific procedures, such as sample types, DNA extraction protocol, number of samples analysed, testing workflow, and result interpretation. Reducing or summarizing the general background (e.g., "the test aims to detect…") could help streamline the section and improve scientific rigour. The statement "cultures were prepared by spreading various samples on slides," followed by Ziehl-Neelsen staining, as written, is misleading. Culture preparation involves growing Mycobacterium tuberculosis on solid or liquid media (e.g., Löwenstein-Jensen or MGIT). Spreading samples on slides followed by Ziehl-Neelsen staining is part of smear microscopy and serves as a follow-up test for post-culture confirmation. Lines 104-106 Figures: Consider revising the sentence to refer to the figures more naturally. Consider expanding the legends in your figures. A good figure legend should be descriptive and informative enough to allow the figure to be understood independently of the main text. DISCUSSION While the discussion contains valuable scientific information, particularly regarding the mechanisms of isoniazid resistance, it tends to focus more on general background knowledge rather than interpreting the specific findings of the present study. As a result, the section feels disconnected from the actual data collected. I recommend restructuring the discussion to better highlight the significance of the 9% isoniazid resistance found in this study. This raises the question: 9%, then what? What are the implications for tuberculosis control in Morocco. How the mutation profile (63% katG vs. 37% inhA) relates to national or global trends. The potential limitations of the study and how they might influence the findings. The discussion opens with a general statement on antibiotic resistance in both TB and malaria. Focusing solely on tuberculosis would make this paragraph more scientifically accurate and better aligned with the manuscript's subject. The phrase "a series of international studies…" is vague and lacks specific references. Please cite key studies or rephrase the sentence to avoid generalisations. Lines 204-205 The statement "isoniazid resistance is a global issue with variable prevalence across regions" is a repetition of the idea in the previous sentence. Consider merging the two sentences to improve flow and avoid redundancy. Line 206 Comparing isoniazid resistance rates across countries without adequate context can be misleading. Prevalence depends on various factors, such as population characteristics, diagnostic methods, and study design. Rather than listing other studies, consider focusing more deeply on why your study found a 9% resistance rate, and what this means within the Moroccan context. For example: Could regional diagnostic gaps contribute to under- or overestimation? Are there programmatic issues that could explain the mutation patterns observed? Lines 208-217 CONCLUSION This conclusion does not reflect the main objective of the study. Instead of summarising and interpreting the findings related to isoniazid resistance, it focuses broadly on tuberculosis and drug resistance in general. There is no mention of the 9% isoniazid resistance prevalence found, nor of the katG and inhA mutation patterns observed. The conclusion should directly address these results and explain their implications for tuberculosis control, diagnosis, and treatment, particularly within the Moroccan context. I recommend revising the conclusion to clearly reflect the study's specific contributions and to highlight the relevance of its findings.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Not at all

    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

  4. Version published to 10.1099/acmi.0.000928.v2 on Access Microbiology
  5. Access Microbiology

    Comments to Author

    Amri et al submit a report on a retrospective study of the prevalence of isoniazid resistance in Mycobacterium tuberculosis complex isolates from the Mohammed V Military Instruction Hospital. The paper is for the most part well written but could include some further expanding of results and clarification. I have the following comments: 1. The abstract should mention why the authors undertook this study (IE data on resistance in Morocco is lacking). 2. The introduction in its current form could be expanded. I am left wanting more information. a. Is there a reason younger individuals are more prone to TB in Morocco? b. How widespread is drug resistance/multi drug resistance in Morocco? c. What have the other studies on INH resistance found? And potentially what are their limitations? (IE short time period examined, only focus on pulmonary and not extrapulmonary, etc) d. Is Morocco using the BCG vaccine and how widespread are HIV/TB co-infections in Morocco? 3. Materials and methods: Explain the types of statistical analysis that were performed. 4. A Table incorporating patient data (age, sex, type of TB, known drugs treated with) and sample collection site for each patient to supplement the text and Figure 1 would be informational to readers and easier to follow than just the text. 5. If males make up 74% of patients, it is not surprising that more men had INH resistant TB than women (It would be surprising if women had more INH resistant TB, potentially signaling something specific to gender or sex. I would like to see of the 74% men how many had INH resistance and of the 26% women how many had INH resistance? 6. Figure 2: Define "S" and "R" as susceptible and resistance. 7. Figure 3: Denote gene names correctly: inhA should be italicized, katG should be lowercase "K" and italicized. If talking about the protein: denote as InhA and KatG. Please note that gene names should be italicized with the first letter lowercase in all instances found in the paper. 8. Lines 187-189: The reference cited makes no mention of MDP1 or dormant bacteria. This makes me question if other references are correct. Additionally, I can't find any reference cited that mentions MDP1 conferring INH resistance. There is a paper I found (doi: https://doi.org/10.1074/jbc.M111.333385) referencing MDP1 as a katG regulator in BCG but it is unclear how translatable this would be to clinical isolates, as MDP1 appears essential and the phenotype described was due to conditional knockdown and not altered function. a. Dormancy and persister cells are a unique phenomenon that likely renders cells tolerant due to suppressed or altered metabolic states. The authors even use a robust review citing this information later in the discussion (citation #12). 9. Lines 192-196: Make sure to differentiate that the katG mutation prevalence referred here is from a global study (as based on earlier, S315 mutations are 60-70% in Morocco) and cite this information. 10. What is the MIC of katG mutations to compare to inhA? 11. The second half of the discussion is a nice summary of katG and inhA INH resistance mutations and alternative routes to resistance, but I fail to see the how this information ties into the current study unless the authors did work identifying if isolates had any efflux gene mutations as well. Is this a limitation of the study? It might be worth mentioning that phenotypically resistant isolates may be katG/inhA mutation negative due to alternative routes?

    Please rate the manuscript for methodological rigour

    Good

    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

  6. Access Microbiology

    The reviewers have highlighted major concerns with the work presented. Please ensure that you address each of their comments and make corresponding changes to the manuscript and figures. In addition to the reviewer comments, several other points need to be addressed for this manuscript to be considered fit for publication: - The ethical approval for this study is unclear. While the use of patient samples may mean that direct consent is not necessary, IRB/institutional approval is still required to provide assurance that ethical guidelines were followed throughout the study. An IRB protocol or signed approval form would be sufficient, along with an updated statement in the text. - The included figures are low-quality and lack accompanying legends. Please ensure that all figures are generated in a high-quality format and have legends describing what they show - There is a concerning lack of references used in the manuscript, particularly in the introduction. Please be sure that factual statements have relevant sources cited in the text.

  7. Access Microbiology

    Comments to Author

    This manuscript, "Genotypic study of isolated resistance to isoniazid in the Mycobacterium tuberculosis complex in a Moroccan hospital," is a study that provides important insights into the genetic basis of isoniazid resistance in Morocco, a topic of significant public health importance. Below are my comments and suggestions to enhance the quality and impact of your manuscript. Major Comments: - While the manuscript identifies the prevalence of katG and inhA mutations, it does not sufficiently explore the clinical implications of these findings. Please expand on how these mutations may influence treatment strategies or guide local tuberculosis (TB) control programs. Consider discussing the potential role of alternative therapeutic options, such as ethionamide, especially in cases with inhA mutations. Consider elaborating on how these mutations affect isoniazid activation and efficacy, referencing broader literature on the topic. - The study mentions global and national resistance trends but lacks a detailed comparison with similar studies from other countries in the region. Adding such comparisons could provide better contextualization of your findings. - The manuscript primarily relies on basic descriptive statistics. Incorporating advanced statistical tools, such as significance testing or confidence intervals for prevalence rates, would strengthen the reliability of your conclusions. Specifically, address whether the sample size (464 patients) is representative of the TB population in Morocco and whether any sampling biases could have influenced the results. - Provide additional details about the quality control measures implemented during molecular testing, beyond the use of H37Rv as a reference strain. Also, clarify how the GenoType MTBDRplus assay results were validated, if applicable. - The figures are poorly executed and unrepresentative. Use a professional tool for producing publication-quality figures. Also, include additional details in the figure legends to make them standalone and more informative. - The manuscript does not provide clear future research directions or practical recommendations. Including these could enhance the utility of your findings for both researchers and public health practitioners.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Poor

    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?

    No: no specific ethical approval number is mentioned, there are no apparent ethical violations

  8. Version published to 10.1099/acmi.0.000928.v1 on Access Microbiology