Genetic diversities of Mycobacterium tuberculosis complex species in Western Kenya

  1. Charles Komen Chelimo
  2. Paul Oyieng Angienda
  3. Charles Ochieng Olwal
  4. Henry Nyamogoba

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Abstract

Background. Tuberculosis (TB) remains a high-burden infectious disease worldwide. Mycobacterium tuberculosis complex (MTBC) is the aetiological agent of TB.

Research Gap. The TB burden is significantly linked to the development of drug-resistant strains. Thus, there is an urgent need for close surveillance of MTBC circulating in a given region, such as Western Kenya, for treatment of TB.

Aim. To determine the proportion of MTBC species, strains and genetic diversities in circulation in HIV/AIDS-prevalent regions, and Western Kenya in particular. The clinical MTBC isolates were collected from Moi Teaching and Referral Hospital (MTRH) at Eldoret-Kenya during 2013–14. All clinical MTBC isolates were confirmed by the gold standard method (Löwenstein–Jensen medium culture) before inclusion in the investigation.

Methodology. Twelve-loci mycobacterium interspersed repetitive unit – variable-number tandem repeats (MIRU-VNTR) genotyping was performed to determine the circulating species/strains of MTBC using the www.miru-vntrplus.org web platform. Allelic diversity was calculated using the Hunter–Gaston diversity index (HGDI).

Results. The species M. tuberculosis , Mycobacterium bovis , Mycobacterium africanum , Mycobacterium pinnipedii , Mycobacterium microti , Mycobacterium caprae and Mycobacterium canetti were identified in the MTBC population. These strains were found in the Beijing, Latin American Mediterranean, Uganda 1/2, East African Indian, Ilama, West African 1/2, Harlem, URAL, Ghana, Seal, Cameroon and Vole etc. regions of Western Kenya. Notably, some isolates had unknown (new/unassigned) species. The strains were grouped into nine clusters with a clustering rate of 31.18 % and a high allelic diversity index of 0.53 was observed.

Conclusion. The present findings suggest that there is an urgent need for more awareness among healthcare professionals and stakeholders concerning the existence of foreign MTBC species/strains in Kenya. Furthermore, 12-loci MIRU-VNTR may not be suitable for the surveillance of MTBC strains in circulation in Kenya. Thus, high-resolution techniques such as whole-genome sequencing need to be adopted to resolve the genetic diversity and establish evolutionary trends for future and archived samples. This knowledge will be crucial in restraining TB, providing insights into new drug development, and developing prevention, control and treatment strategies for TB.

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  1. Version published to 10.1099/acmi.0.000729.v3 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000729.v2 on Access Microbiology
  4. Access Microbiology

    Thank you for your submission. I have selected minor amendments as my decision for the manuscript. Please address reviewers comments and return the manuscript. Note: "Reviewer 2 - Comments 9: How are your results of study different from other studies on MTBC and NTM, which are already published in various international journals?" Editor comment - While this will add value (so please address where possible), ACMI accepts reproduction studies, so novelty is not an acceptance criteria.

  5. Access Microbiology

    Comments to Author

    First of all, this study is an informative study on Genetic diversity of Mycobacterium tuberculosis complex but it needs to improve grammatical pitfalls in some points in the manuscript and further add 4-5 latest references in the present study, which are correlated with the present data. Comments 1: The title of manuscript does not provide accurate information and it will be modified as: "Mycobacterium tuberculosis complex species diversities in Western Kenya" Or "Genetic diversities of Mycobacterium tuberculosis complex species in Western Kenya" Or "A genetic diversity of Mycobacterium tuberculosis complex species" Author response: Comments 2: Lines 9-33, in abstract paragraph; some mistakes and I have corrected the sentence and you have modified your abstract section as: Background: Tuberculosis (TB) remains a high burden infectious disease worldwide. Mycobacterium tuberculosis complex (MTBC) is the etiological agent of TB. Research gap: TB burden is majorly linked to development of drug resistant strains thus there is urgent need for close surveillance of MTBC circulating in a given regions like Western Kenya for the treatment of TB. Aim: To determine the proportion of MTBC species, strains and genetic diversities in circulation in an AIDS/HIV prevalent regions especially Western Kenya. The clinical MTBC isolates were collected from Moi Teaching and Referral Hospital (MTRH) at Eldoret-Kenya during 2013-14. All clinical MTBC isolates were confirmed by the gold standard method (i.e. Lowenstein-Jensen medium culture) and further used in investigation. Methodology: A 12-loci of Mycobacterium Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR) genotyping was performed to determine the circulating species/strains of MTBC using www.MIRU-VNTRplus web platform. Allelic diversity was calculated using the Hunters-Gaston Diversity Index (HGDI). Results: The species M. tuberculosis, M. bovis, M. africanum, M. pinnipedii, M. microti, M. caprae and M. canetti were identified in the MTBC population. These strains were found in the Beijing, Latin American Mediterranean, Uganda 1/2, East African Indian, Ilama, West African 1/2, Harlem, URAL, Ghana, Seal, Cameroon and Vole etc. regions of Western Kenya. Notably, some isolates had unknown (new/unassigned) species. The strains were grouped into nine clusters with a clustering rate of 31.18% and a high allelic diversity index of 0.53 was observed. Conclusion: The present findings suggest that there is an urgent need for more awareness among healthcare and stakeholders on the existence of foreign MTBC species/strains in Kenya. Furthermore, 12-loci MIRU-VNTR may not be suitable for the surveillance of MTBC strains in the circulation in Kenya. Thus, high-resolution techniques i.e. Whole Genome Sequencing needs to be adopted to resolve the genetic diversity to establish evolutionary trends for future and archived samples. This knowledge will be crucial in restraining TB, giving insights into new drug development, and prevention, control/treatment strategies of TB. Line 34: Write the keywords (3-5 words) accurately and alphabetically order as Genetic Diversity Hunters-Gaston Diversity Index (HGDI) Mycobacterium Interspersed Repetitive Units (MIRU) Mycobacterium tuberculosis complex (MTBC) Tuberculosis (TB) Variable Number Tandem Repeats (VNTR) Author response: Comments 3: Lines 35-44, introduction first paragraph; Approximately 10.6 million individuals contracted tuberculosis (TB) (abbreviated first time then abbreviated form write at next time), a slight increase from the 10.1 million cases reported in 2020. Additionally, the number of TB-related deaths in 2021 was ~1.6 million, including 187,000 people with AIDS/HIV, compared to 1.5 million deaths in 2020, which included 214,000 individuals with AIDS/HIV (Add WHO reference). Furthermore, there was a 3.6% rise in the TB-incidence rate in 2021 compared to 2020, marking a departure from the nearly 2% annual decrease observed over the past two decades [1]. Moreover, approximately 23% of the world's population has latent TB infection, and could develop active TB in their lifetime [2]. The African region plays a pivotal role in the global TB-epidemiological context [3]. Kenya is among the high TB prevalent countries in the world and reports substantial TB-related mortality [4]. Where is the data of TB after COVID-19 pandemic? Discuss some latest research articles (as mentioned below) in your introduction parts. Add a small paragraph here as. TB is a major public health concern worldwide, ranking above HIV/AIDS and its diagnosis exhibits serious challenges owing to paucibacillary nature of specimens and localization of disease at sites that are difficult to access (Khan et al., 2022; Kamra et al., 2022). Until the COVID-19 pandemic, TB was the foremost cause of death from a single infectious agent and is now the second leading infectious killer after COVID-19 (Kamra et al., 2023). Kamra et al., (2022). Diagnosis of genitourinary tuberculosis by loop-mediated isothermal amplification based on SYBR Green I dye reaction. Biotechniques 73(1): 47-57. https://doi.org/10.2144/btn-2022-0027. Khan et al., (2022). Diagnosis of osteoarticular tuberculosis by immuno‐PCR assay based on mycobacterial antigen 85 complex detection. Letters in Applied Microbiology 74(1): 17-26. https://doi.org/10.1111/lam.13567. Author response: Comments 4: Which proteins are common in the MTBC species like M. bovis bacillus Calmette-Guerin (BCG) and nontuberculous mycobacteria (NTM)? (Add a few 1-2 lines on these proteins and hints taken from Khan et al. 2020 research article and cited in your manuscript). Khan et al., (2022). Diagnosis of osteoarticular tuberculosis by immuno‐PCR assay based on mycobacterial antigen 85 complex detection. Letters in Applied Microbiology 74(1): 17-26. https://doi.org/10.1111/lam.13567 Author response: Comments 5: Lines 45-50, introduction part; second paragraph: Mycobacterium tuberculosis is the main causative agent for TB [5]. There are eight species within the M. tuberculosis complex (MTBC), namely M. tuberculosis, M. bovis, M. africanum, Bacillus Calmette-Guerin (BCG), M. microti, M. carprae, M. pinnipedii and M. cannettii [6]. Although most pulmonary tuberculosis is mainly attributed to M. tuberculosis, there is need for surveillance of species and strains of MTBC in circulation to inform on suitable drugs to minimize development of anti-TB drug resistance [7]. Write a brief paragraph on nontuberculous mycobacteria (NTM)? Author response: Comments 6: Which MTBC specific regions of difference encoded proteins are absent in the majority of M. bovis bacille Calmette Guerin (BCG) substrains and non-tuberculous mycobacteria? Dahiya et al., (2020). Diagnosis of tuberculosis by nanoparticle-based immuno-PCR assay based on mycobacterial MPT64 and CFP-10 detection. Nanomedicine 15(26):2609-2624. https://doi.org/10.2217/nnm-2020-0258. Author response: Comments 7: What is the difference between MTBC and nontuberculous mycobacteria (NTM)? Author response: Comments 8: Lines 47-49: Although most pulmonary tuberculosis is mainly attributed to M. tuberculosis, there is need for surveillance of species and strains of MTBC in circulation to inform on suitable drugs to minimize development of anti-TB drug resistance [7]. Add a few lines on Pulmonary TB and Extra-pulmonary TB? Hints taken from this reference: Khan et al. (2021). Diagnosis of osteoarticular tuberculosis: multi-targeted loop-mediated isothermal amplification assay versus multiplex PCR. Future microbiology 16(13):935-48. https://doi.org/10.2217/fmb-2021-0030. Author response: Comments 9: How are your results of study different from other studies on MTBC and NTM, which are already published in various international journals? Author response: Comments 10: What is the limitation of your present study? Author response:

    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?

    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

    Location and Study Design: It was stated that the research was conducted at Moi Training and Referral Hospital and that this hospital serves the entire Western Kenya community.It was announced that the study was retrospective and that samples archived between 2013-2014 were used.It was stated that the selection criteria were clear, stating that only samples determined to be TB positive in the Lowenstein-Jensen environment were used. However, the criteria for concluding 'no contamination' were not disclosed; This needs to be clarified in terms of methodology. Sample Processing: Details are provided on how the samples were processed, but it would be helpful to provide more information on specific laboratory protocols and storage conditions of the samples. Additionally, how samples were selected and the criteria by which they were classified as TB positive could be explained in more detail. MIRU-VNTR Typing and Determination of Allelic Diversity: It was stated that MIRU-VNTR typing was performed using 12 different loci and the primer sequences used were previously published. It was stated that this typing was made through the MIRU-VNTRplus database. Although it has been stated that the methods have been detailed in previous studies, it would be good to give a brief explanation here so that the reader can quickly understand these methods. Cladogram and Clonal Complexes: A neighbor-joining dendrogram was constructed and clonal complexes were identified using the minimum tree. It would be useful to add more methodological details about how these analyzes were performed. General evaluation: The methodology is generally well explained, but more detail is needed in some areas. In particular, a more detailed description of how contamination is assessed, sample processing protocols, and analysis methods would strengthen the methodology. Ethical approval and sample use are clearly stated, increasing the reliability of the study. However, it would be useful to include details of previous studies referenced here so that the reader can fully understand the entire methodology. DISCUSSION Further discussion of the limitations of the MIRU-VNTR analysis and generalization of the results would have been helpful.Additionally, discussing the concrete implications of the findings on TB management and control strategies may further increase the impact of the study 1 western-------Western 47 carprae---------caprae 47 cannettii---------canetti 76 western---------Western In general, although the work is done properly, there is no originality. It would be better to enrich the article by adding different data and perhaps statistical studies.

    Please rate the manuscript for methodological rigour

    Poor

    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?

    Yes

  7. Version published to 10.1099/acmi.0.000729.v1 on Access Microbiology