Tuberculosis (TB) is the main cause of morbidity and mortality, due to a single infectious cause, globally, with over 10.0 million new cases and 1.3 million deaths annually, a quarter of all cases coming from Africa [1]. Cameroon is among the top 20 countries with the highest burden, with 46,000 new cases annually and an incidence rate of 150/100,000 per year [1]. TB disease, is due to infection with Mycobacteriumtuberculosis complex (MTBC) [2], comprising nine different lineages; lineages 1 - 4, and 7 referred to as M. tuberculosissensustricto, lineage 5 and 6 as M. africanum lineages, lineage 8 recently discovered in the Great Lakes region of East Africa [3] and lineage 9 in East Africa [4]. Lineages 2, 3 and 4 have a global distribution (generalists), whereas other lineages are more geographically restricted (specialists), like L7, which is restricted to the Horn of Africa, L5 and L6, are found mostly in West Africa [3][5][6]. Lineage 2 (the Beijing genotype) originated in East Asia [7] and has expanded to some parts of the world, including Africa [8]. Lineage 4 (the Euro-American lineage) is globally distributed, though some of its genotypes (such as the Uganda and the Cameroon genotypes predominant in Ugandan and Cameroonian populations, respectively) are geographically restricted. Despite control efforts, there is evidence of ongoing TB transmission in Cameroon [9]. Early studies reported Mycobacteriumafricanum West, as the leading etiology of pulmonary TB in Cameroon [10]. However, recent studies suggest the predominance of M. tuberculosis in place of M. africanum [11]-[13]. The L4 LAM_10 Cameroon genotype has a high clustering rate, indicating active transmission and expansion within communities and this is often linked to socio-economic factors, healthcare access, and population density, coinciding with human population growth and movement, particularly in regions with limited tuberculosis control measures. The emergence and spread of MTBC genotypes previously absent in a region could be associated with immigration, clinical and demographic factors, or evolution of MTB isolates, due to evolutionary pressures such as drug resistance, host adaptation (host-pathogen interaction) and environmental factors, suggesting a constant variation in MTBC genotypic population structure [14][15]. Furthermore, in Cameroon, previous studies on TBMTB were largely based on conventional phenotypic and genotypic methods, such as Restriction Fragment Length Polymorphism (REFLP), Spoligotyping and Mycobacterium Intersperse Repetitive Unit-Variable Number (MIRU-VNTR), with subjective interpretations often yielding inconclusive results [16], limited ability for MTBC sub-lineage resolution [17] and unable to discriminate between MTBC and Non-Tuberculous Mycobacteria (NTM) genotypes. To expand these findings, Whole Genome Sequencing (WGS) for higher phylogenetic resolution was used to map the genetic diversity of MTB and their drug-resistance patterns, in pulmonary TB patients from the Southwest and Littoral Regions of Cameroon.

In this study, we report the genetic diversity and drug-resistance patterns of MTB pulmonary TB isolates using whole genome sequencing. A total of 125 MTB DNA samples were sequenced from which 88 high-quality sequence reads were obtained for analysis. The TB severity was determined following the TBScore II algorithm (modified BANDIM score). Bacteriological analysis (AFB microscopy), showed that majority of participants, 43/125 (34.4%), had an AFB smear grade of 3+. It has been shown that effective sputum smear conversion is hindered by initial high bacillary count (3+) and advancing age of TB patients [26]. Smear microscopy has also been reported to reflect the extent of disease severity, with higher smear grades, associated with extensive lung damage and cavitation. However, reports indicate that close to 2% of patients may be smear negative at treatment completion and this could be due to non-viable bacteria and colonization by non-tuberculous mycobacteria [27]. Early diagnosis is paramount to interrupting TB disease transmission. It is established that AFB smear-positive patients are the primary source of TB transmission to healthy individuals if left untreated. Studies have reported that close to 17% of TB transmission is by AFB smear-negative TB suspects; hence, the risk of disease transmission by AFB-negative cases to healthy individuals cannot be ignored [28].

Africa is home to extensive genetic diversity of humans and pathogens, including mycobacteria, and it is the only continent with all known MTB lineages. Cameroon, a country situated in the West and Central parts of Africa, is endemic for TB and there is still a dearth of information on MTBC population diversity in the country. This can be important for the development of better diagnostic tools and novel vaccine therapies, and to understand the changing structure of the tuberculosis epidemic in Cameroon. Epidemiological studies on MTB in Cameroon have previously been based on phenotypic characterization and low-resolution molecular typing methods. In this study, WGS approach was used to genotype MTBC in newly acquired pulmonary tuberculosis cases, seeking care in TB treatment centers in the Southwest and Littoral regions. The analysis revealed a predominance of MTB lineage 4, accounting for 85/87 (98%) of the isolates, whereas the East African Indian lineage (Lineage 3) and the East Asian lineage (Beijing Ancestral 3) were less prevalent, accounting for 1/87 (1%) each of all isolates. No Indo-Oceanic lineage (lineage 1) strains were identified. This observation is in line with the findings of Negrete-Paz and colleagues, who suggested that lineage 4 and Beijing isolates were significantly associated with pulmonary TB, whereas lineage 1 and the ancient Beijing genotype were significantly associated with extra-pulmonary TB [29]. Thus, we may have missed these lineages in our study, which focused on pulmonary TB. The modern lineages, 2, 3 and 4, are more virulent, genetically more diverse, and have a global geographic distribution [30] compared with the ancient lineages, Lineages 1, 5, 6, 7, 8 and 9, which are less virulent and geographically restricted [31]. Modern lineages are associated with higher inflammatory response, than ancient strains, and this has resulted in a selective advantage of the modern lineages, leading to impaired bacterial control by the host, rapid disease progression, and heightened disease transmission [32]. Studies in the early 1970s by Huet and colleagues in the West Province of Cameroon found M. africanum (L5 and L6) to be the main etiologic agent of TB, with a prevalence rate of 56 %. However, this paradigm has shifted over the last five decades. Studies from early 2000 to the present day show a consistent regression of M. africanum and the predominance of M. tuberculosis as the etiologic agent of TB in Cameroon, with a drastic drop in the M. africanum prevalence from 56% [12] to 6% [16], to almost zero. M. africanum genotypes and M. bovis were not identified in this study. This finding further confirms the decline of M. africanum, and the rarity of M. bovis in human TB infection in Cameroon. All the Mycobacterium isolates identified in this study were genotypes of M. tuberculosis. This is consistent with previous findings, in which M. tuberculosis accounted for more than 90% of TB cases [33][34]. This raises the question of why M. africanum is regressing in Cameroon, while it remains high in most Central and West African countries such as Benin [35][36], Mali [37] and the Gambia [38]. One hypothesis is that the systematic introduction of newborn BCG vaccination in Cameroon may have selected for some genotypes, as with the case of the Beijing family (in East and Central Asia) [39][40]. Hence, M. africanum may have diminished significantly as the cause of TB after the widespread use of BCG vaccination in Cameroon. However, the low sample size may constitute a bias, accounting for the absence of M. africanum and M. bovis in our study. LAM-10 Cameroon sub-lineage of lineage 4 accounted for 42/83 (42%) of all sub-lineages in our study and is the genotype that is the leading cause of TB in Cameroon. It is hypothesized that the Cameroon family has recently emerged through clonal expansion and is heterogeneously distributed in the population. The LAM_10 Cameroon strain, as suggested by similar studies on the Beijing family, could likely have a selective advantage over other M. tuberculosis genotypes in Cameroon, in terms of virulence, pathogenesis, and epidemiologic characteristics. Phylogenetic dating suggests that the Cameroon genotype diverged from a common ancestor approximately 161 years ago to form three main clusters [41]. A limitation of this study is that a transmission cluster analysis was not performed to determine the transmission pattern and cluster rate of the LAM_10 Cameron strain in the study population. The data revealed the L4 sub-lineages Haarlem (20%), the LAM (1%), and the Uganda II (2%) sub-lineages and the Delhi/CAS (1%), a sub-lineage of lineage 3, all of which have been reported previously in Cameroon [16]. Of interest is the Beijing ancestral 3 genotype (L 2.2.1), belonging to lineage 2, not previously reported in Cameroon. The absence of this genotype in previous studies could be explained by the inability of the genotyping methods used to identify lineage 2 (Beijing-type) sub-lineage-specific loci [18] or by the rarity of this genotype in West Africa [42]. We also identified the Coll lineage 4.7, previously described by Malm and colleagues in 2017, as a new Euro-American sub-lineage in Congo Brazzaville and named it the Congo type [43]. The emergence in our study of geographically more restricted isolates like Uganda II, predominant in Uganda, East Africa [44]; the Congo genotype (Coll lineage 4.7) in Congo Brazzaville and the Beijing ancestral 3 genotype in East and Central Asia [45][46], suggests cross-border transmission through migration, followed by local spread. This warrants close monitoring and investigation of these strains in the overall transmission dynamics and epidemiology of TB in Cameroon. The introduction of new MTB strains that are more transmissible, more virulent, and more prone to drug-resistance has been associated with migration, especially population movement, to larger cities in search of better health care facilities and employment prospects and can drive the current changing TB situation [47]. In the current context of globalization and population movement, it is a challenge to ascertain and characterize the mycobacterium strains in a given human population and to control the emergence of drug-resistant strains and their dissemination. In addition, this study revealed significant association between TB disease severity and HIV positivity and dyspnea. In high-burden settings, HIV co-infection is the most important risk factor for developing active TB [48]. People living with HIV (PLHIV) are at a much higher risk of developing active TB due to their weakened immune systems. The risk of TB increases as the CD4 cell count decreases, making PLHIV particularly vulnerable. Severe TB primarily affects the lungs, causing inflammation and damage to lung tissues. This can lead to airflow obstruction and reduced lung function, resulting in dyspnea [49]. Studies have shown that the degree of dyspnea is related to the functional capacity of patients with TB sequelae. This means that more severe dyspnea is often associated with greater limitations in physical activity and overall lung function [50]. Upper and higher income earners have lower odds of developing severe TB compared to middle to low-income earners. TB is a disease of poverty; a lack of basic health services, poor nutrition, and inadequate living conditions all contribute to the spread of TB and its impact upon the community. An absence of good-quality health care facilities is common in poor communities. With no health services to diagnose or treat patients, there is a longer delay between disease and cure, perpetuating the spread of TB (StopTB.org). Our study showed that the LAM_10 Cameroon was significantly associated with moderate TB (OR = 7.94, p = 0.03) and this could imply a more severe disease presentation although our data could not establish this association. No direct correlation has been established consistently between the LAM_10 genotype, drug resistance and HIV positivity, as shown in this study. However, the lack of correlation in this study could be attributed to low statistical power, resulting from the small sample size. Although the LAM_10 genotype is associated with Majority of TB cases, TB disease severity classification is typically based on radiographic findings, bacillary load, and clinical symptoms and not just MTB genotype or strain.

We used WGS to infer drug-resistance to first- and second-line anti-TB drugs in 88 MTB clinical isolates from newly acquired TB cases. 79 (89.8%) of the isolates were sensitive, whereas 9 (10.2%) were drug-resistant. This finding is highly suggestive of circulating drug-resistant TB. One isolate of the Haarlem sub-lineage was isoniazid mono-resistant, and three isolates, one Euro-American and two LAM_10 Cameroon, were Rifampicin resistant, and one (mixed isolate) was resistant to Pyrazinamide (PZA). No Multi-Drug-Resistant (MDR) isolates were identified. We found Rifampicin, isoniazid, and streptomycin resistance rates to be 3.4%, 1.1% and 4.5% respectively, which are far less compared to the rates of 27.6%, 54.1% and 25.5% for the same drugs, identified by using Drug Susceptibility test (DST) [51]. The difference could be attributed to sampling bias or inherent variability of the DST compared to WGS approach in this study. The trends in drug resistance in this study are similar to those reported by Noeske and colleagues in 2018 [52] and Thumamo etal. 2019 [53], who found Rifampicin resistance rates to be 1.6% and 8.8% respectively in the Littoral Region of Cameroon, using GeneXpert/RIF. These findings suggest a decreasing trend in drug resistance to first-line anti-TB drugs. This could be explained in part by a scale-up in national TB control measures such as improved TB diagnosis, proper adherence to TB treatment regimens owing to the Directly Observed Treatment (DOT) program, effective follow-up of TB patients and contact tracing, and regular surveillance and monitoring of Rifampicin resistance or MDR-TB cases.

Rifampicin resistance is acquired through several different mutations in the rpoB gene. We also identified a double Rifampicin resistance mutation, rpoB_His445Asn and rpoB_Ser441Ala, in a poly-resistant Euro-American sub-lineage 4.1.3 and rpoB_His445Asn and rpoB_Pro454Ser in two LAM-10 isolates each. The rpoB_His445Asn and rpoB_Ser441Ala are positive mutations primarily occurring within the Rifampicin Resistance-Determining-Regions (RRDRs) and have been reported in several studies [54]. The rpoB gene in MTBC encodes the beta subunit of RNA polymerase, which is essential for RNA transcription [55]. It is known that more than 95% of Rifampicin-resistant strains harbor a mutation within an 81-bp region of the rpoB gene between codons 507 and 533 and this region is called the Rifampicin Resistance-Determining-Region (RRDR) [56]. The highest level of Rifampicin resistance of the rpoB gene occurs in codons 531 and 526. The mutations identified in this study are very low-level mutations that occur outside the RRDR [57]. The poly-resistant isolate, in addition to having the ropB gene mutation, also harbored the canonical mutation rrs1402C>A in the “hot” rrs gene, reported to be associated with cross-resistance to the second-line aminoglycoside anti-TB drugs amikacin, capreomycin and kanamycin [58]. However, it is clear from previous studies that mutations in rrs gene are not always associated with amikacin resistance; traditional drug susceptibility testing will still be helpful to evaluate such samples [59]. The rrs gene encoded the Mycobacterium 16S rRNA, which plays a crucial role in protein synthesis. The isoniazid-resistant isolates we identified in this study acquired the fabG1inhA promoter-region mutation, known to confer resistance to isoniazid and ethionamide. Isoniazid resistance is linked to several gene mutations, such as katG (catalase-peroxidase gene), kasA (β-keto-acyl-АСР-synthase gene), ndh (NADH-dehydrogenase), inhA region (NADH-dependent ACP-reductase InhA gene) and ahpC region (alkyl-hydroxyperoxide-reductase gene). katG and inhA gene mutations are most clinically relevant and determine resistance in most of the clinical isolates [60]. The fabG1inhA gene codes for the Mycobacterium 3-oxoacyl-thioester reductase, an enzyme which has a crucial role in mycobacterium mycolic fatty acid biosynthesis [61] essential in bacterial cell wall synthesis, and isoniazid is known to interfere with this process by targeting mycolic acid [62].

Of the 4/88 (4.5%) isolates that were resistant to streptomycin, we found the mutation rpsL_Lys43Arg in rpsL gene of a Beijing strain (2.2.1). We also identified the gid_351delG in the SM/gid gene of a Euro-American (4.1), gid_115delC and gid_386delG in the SM/gid genes of 2 Harlem genotypes. Mutations in the rpsL gene (especially rpsL_Lys43Arg) account for approximately 80% of streptomycin resistance [63][64]. However, these reports are at variance with our findings since we identified only 1/88 (1.1%) of the isolates harbored the rpsL gene mutation. Rather, we identified a mutation in the SM/gid gene in three isolates. Negrete-Paz and colleagues reported the gid_351delG mutation associated with resistance to streptomycin in extra-pulmonary TB patients. The gid gene in mycobacteria encodes a tRNA methyltransferase, which is responsible for adding a methyl group to the 2’-O position of the guanosine at the 18th nucleotide of tRNAs. This modification is essential for proper tRNA function and stability.

One of the isolates identified as a mixed, harbored the rare mutation panDVal138Ala in the panD gene conferring resistance to pyrazinamide. Zhang and colleagues reported the panDVal138Ala mutation in M. canettiandM. tuberculosis as a potential new mutation conferring resistance to PZA, besides the well-known pncA and rpsA mutations [65]. This finding is similar to that of Özgür etal., in 2022 [66], who found one Mtb strain out of 11 with the panD mutation. The panD gene in Mycobacterium, encodes aspartate decarboxylase, a 139 amino acid (15 kD) enzyme involved in several essential processes, such as the formation of β-alanine from l-aspartate, which is a precursor for pantothenate (vitamin B5) and coenzyme A (CoA) biosynthesis. CoA is crucial for energy metabolism, where carbohydrates, fats, and proteins are utilized. The exact activity of Pyrazinamide is not known; however Zhang and colleagues speculated that Pyrazinamide might inhibit pantothenate and CoA synthesis, thereby interfering with diverse metabolic functions such as energy production and fatty acid metabolism in M. tuberculosis. PanD mutation in M. tuberculosis has been shown to cause higher attenuation of virulence in mice than the BCG vaccine, indicating that the panD may be critical for survival and persistence of the bacilli in vivo [67]. The absence of the panD gene in humans and its significant role in the cellular metabolism of Mtb make it a potential drug target candidate. The mutations Ser441Ala, His445Asp and Pro454Ser in the rpoB gene, the 386 delG, 115 delG and 315 delG in the gid gene, the panDVal138Ala mutation in the panD gene and the 1402 C>A mutation in rrs gene, were not previously reported in Cameroon.

We recruited pulmonary tuberculosis participants and used WGS approach to map the MTBC population structure in the Southwest and Littoral Regions of Cameroon. Findings revealed a diverse population of MTBC, predominated by the Euro-American lineage (L4). The absence of M. africanum (L5/L6) isolates is additional evidence of its continued previously reported regression in favor of the L4 LAM_10 Cameroon genotype as the etiologic agent of pulmonary TB in Cameroon. Drug-resistance profiling revealed new mutations: Ser441Ala, His445Asp and Pro454Ser in the rpoB gene, the 386 delG, 115 delG and 315 delG in the gid gene, the panDVal138Ala in the panD gene and the 1402 C>A mutation in rrs gene. Although RIF mono-resistance as a proxy marker for MDR indicated a low rate of resistance in newly acquired pulmonary TB, there is a need for continuous surveillance of circulating resistant variants in the population. Further investigation would provide additional insight into the genetics, epidemiology, and transmission dynamics of the L4 LAM_10 Cameroon genotype, which may furnish clues to the evolutionary success of modern MTBC lineages.

Not applicable.

All datasets used to generate the present report are available upon reasonable request from the corresponding author and will be deposited into public databases.

This study was supported by the H3Africa Consortium under the project entitled: AnintegratedapproachtounravellingthesusceptibilitytotuberculosisinAfrica, withgrantnumber: H3A/18/003, managed by Science for Africa Foundation (SFA) and Alliance for Accelerating Excellence in Science in Africa (AESA), in partnership with Wellcome Trust (UK), the National Institute of Health (US), and the African Society of Human Genetics (AfSHG) and African Academy of Science (AAS). This work was also supported by DFG, the German Research Foundation (Grant HO 2009/14-1) in the context of the German-African Cooperation Projects in Infectiology (grants awarded to AH, MR, and SW). In addition, AH is funded by the DFG under Germany’s Excellence Strategy—EXC2151—390873048. The funding bodies played no part in the design of the study, collection, analysis and interpretation of data and in writing the manuscript.

Below are the respective contributions of each author in no order: D. N. A., N. T. M., C. M., D. G. K. T.: Co-investigator, led data collection in the field, the laboratory and analysis. D. N. A.: Drafting of the manuscript for publication. F. F. F., A. J. N., M. R., M. J. N., A. A., A. H., E. I. G., S. W.: Co-investigators, participated in the revision of the manuscript. F. N. N.: Generation of the map of study sites. T. M. N., J. F. C.: Co-investigator, supervision of field and laboratory research activities and revision of manuscript for publication. A. G. W., T. D. K., D. H., Y. K. H., B. A.: Co-investigator, supervision of laboratory research activities, and coordination of bioinformatics data analysis. K. B., S. W.: Principal Investigator, in charge of conceptualization and the design of the study, leading and directing research activities and revision of the manuscript for publication. This manuscript was read and approved by all authors involved in this study prior to publication.

Ethical clearance for this study, was obtained from the Cameroon National Ethics Committee, Ref No: 2019/03/1154/CE/CNERSH/SP. The administrative authorization was obtained from the Ministry of Public Health, Ref No: D30-368/AAR/MINSANTE/SG/DROS/NJ. The research authorization was obtained from the Regional Delegation of Public Health for the Littoral, Ref No: 018/AR/MINSANTE/DRSPL/BCASS/CNT. The protocol was reviewed and approved by the Institutional Review Board of the Faculty of Health Sciences, University of Buea. The written and verbal authorizations were obtained from the various TB diagnostic and treatment centres. Informed consent was obtained from participants ≥ 21 years. Assent was obtained for participants between 18 and 20 years and informed consent was obtained from their parent or legal guardian. Confidentiality of each participant’s information was ensured by using individual codes.

The authors would like to acknowledge the staff of the different health facilities that participated in the study, notably the Bonassama District Hospital, New Bell District Hospital, Cite-Sic Catholic Hospital, Logbaba District Hospital, Deido District Hospital, Barcelone Catholic Hospital, Cite des Palmiers District Hospital, Buea Regional Hospital and Limbe Regional Hospital. Additionally, the authors would like to address their gratitude to Mr. Ebwea Mbappe Jacques for facilitating liaison activities between the research team and the different health delegations and health facilities.