Frequency, genetic mechanisms and factors associated with pre-extensively drug-resistant tuberculosis in Northern Nigeria

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This study aims to determine the frequency and mutations associated with fluoroquinolone and aminoglycoside resistance in MTB isolates from patients with pulmonary illnesses in Kaduna State, Nigeria. Materials and Methods A total of 144 MDR-TB-positive isolates previously obtained from sputum samples were collected from 360 individuals and were processed using the NaOH-Na-citrate-NALC method. All isolates were tested for MTB complex using TB Ag MPT64 (SD Bioline), and GenoType MTBDR sl VER 2.0 was used to identify chromosomal mutations in gyrA, gyrB, rrs and eis genes. Risk associated with pre-XDR-TB was assessed using a structured questionnaire, and the generated data were statistically analysed. Results The occurrence rate of pre-XDR in MDR-TB was 24.3% (n = 35) among 9.7% of individuals. Fluoroquinolone resistance was detected in 18/35 (51.4%) due to diverse mutations and missing regions WT1 and WT2 in gyrA (n = 14) and N538D in gyrB (n = 4). The predominant mechanisms of aminoglycoside resistance (41.2%, 7/17) among pre-XDR-TB strains were MUT1 (A1401G) and MUT2 (G1484T) in the rrs gene. Moreover, the MUT1 (C-14T) mutation (23.5%, 4/17) was observed in the eis promoter region. Histories of contact with TB patients (OR = 8.94, 95% CI: 4.1-19.49, p < 0.0001) and prior anti-tuberculosis treatment (OR = 2.36, 95% CI: 1.13–4.94, p = 0.00233) were associated with pre-XDR-TB. Conclusion This study revealed a high occurrence of pre-XDR-TB in the study population and among the MDR strains, which could lead to treatment failures and a higher public health threat. To stop pre-XDR-TB from spreading and growing and to improve treatment outcomes in this and other regions where it is more prevalent, it is imperative to diagnose resistance to second-line anti-tuberculosis quickly before beginning treatment and activate pre-XDR-TB surveillance systems. Tuberculosis rrs eis pre-XDR-TB Drug-resistant TB Nigeria Figures Figure 1 INTRODUCTIONS Rifampicin-resistant Mycobacterium tuberculosis (MTB), often referred to as multidrug-resistant TB (MDR-TB), is a critical priority pathogen and one of the leading 10 causes of infection-associated death, especially in developing countries [ 1 ]. Nigeria is the top in Africa and ranked 6th among the high TB-burdened countries in the world [ 2 ], contributing to 4.5% of the estimated global incidence [ 3 ]. Treating TB is crucial to save lives from this leading infectious killer, prevent severe illness and disability, stop its airborne spread, and combat the dangerous rise of drug-resistant strains by ensuring the bacteria are eliminated, preventing relapse, and protecting the wider community. The standard first-line treatment for drug-susceptible TB involves a two-phase regimen using a combination of four core antibiotics, often remembered by the acronym RIPE (Rifampin, Isoniazid, Pyrazinamide, Ethambutol) [ 4 ]. The need for treating TB with second-line drugs arises when the standard, or first-line, medications are not effective due to drug resistance [ 4 ]. While essential, second-line drugs often require a longer course of treatment (up to two years), can be more toxic, and are associated with frequent adverse events compared to first-line regimens. Therefore, treatment decisions are guided by drug susceptibility testing (DST) to ensure the most effective regimen is selected for each patient, tailored to their specific resistance profile [ 5 , 6 ]. MDR occurs when MTB is resistant to rifampicin (RIF) and isoniazid (INH) with or without other first-line drugs [ 7 ]. In 2023, a global meta-analysis reported that 9% of MDR-TB patients had developed extremely drug-resistant TB (XDR-TB) strains, while 26% had pre-XDR-TB [ 8 ]. XDR-TB is a severe, rare form of TB where the bacterium is resistant to first-line drugs (isoniazid, rifampin) and also to a fluoroquinolone and at least one second-line injectable drug, making it extremely difficult to treat with standard options, leading to longer, more toxic treatments and a higher risk of severe outcomes, especially for people with weakened immune systems such as those caused by human immunodeficiency virus (HIV) [ 9 ]. XDR-TB is defined as MDR-TB that is resistant to any fluoroquinolone and at least one of the three aminoglycoside drugs. Conversely, pre-XDR-TB is defined when the bacterium is resistant to either (a) Isoniazid, rifampin, and a fluoroquinolone or (b) Isoniazid, rifampin, and a second-line injectable (amikacin, capreomycin, and kanamycin) [ 10 ]. Fluoroquinolones (FLQs) and aminoglycosides (AMGs) have long been used as anti-tuberculosis drugs, and their widespread use has led to the development of resistance in clinical isolates [ 11 ]. FLQ resistance in TB is primarily driven by mutations in the quinolone resistance-determining region (QRDR) of gyrA (subunit A) and, less commonly, gyrB (subunit B), which encode DNA gyrase, the enzyme targeted by these essential second-line anti-TB drugs, with gyrA mutations (especially at codons 88–94) being most frequent and conferring higher resistance [ 11 ]. Mutations in the rrs and overexpression of the eis genes are the primary mechanisms responsible for aminoglycoside resistance in MTB [ 12 ]. The rrs gene mutations in the 16S ribosomal RNA gene alter the drug's ribosomal target site, preventing effective binding and conferring high-level resistance to amikacin and kanamycin and moderate resistance to capreomycin [ 12 ]. The A1401G mutation is the most frequent mutation associated with high-level resistance to amikacin and kanamycin [ 12 ]. Mutations or overexpression of the eis gene promoter increase expression of the eis gene, producing more of the Eis enzyme. This enzyme inactivates aminoglycosides by acetylating them (adding an acetyl group), preventing the drug from inhibiting protein synthesis. This mechanism typically confers a low-level resistance to kanamycin but does not usually cause clinically significant resistance to amikacin [ 13 , 14 ]. To prioritise and facilitate the identification of pre-XDR-TB and XDR-TB, several molecular testing platforms, such as GenoType MTBDRsl VER 2.0, have improved the detection and management of drug-resistant TB [ 13 ]. This study aims to determine the frequency and mutations associated with fluoroquinolone and aminoglycoside resistance in MTB isolates from patients with pulmonary illnesses in Kaduna State, Nigeria. MATERIALS AND METHODS Study area and population One hundred and forty-four (144) frozen sputum samples containing MDR-TB previously collected from people with pulmonary illnesses [ 15 ] were thawed and re-analysed at the National Tuberculosis and Leprosy Training Centre (NTLTC), Zaria, Nigeria. The samples were decontaminated using NaOH-Na-Citrate-NALC method. An inoculum of 0.1mL of decontaminated sputum sample was prepared and incubated at 37°C for 8 weeks. Colonies from the positive culture slants were used to identify the MTB complex using rapid immunochromatographic test kits. Study design and data collection This hospital-based surveillance study included individuals seeking healthcare at NTLTC, regardless of age or sex. Data from this study were collected using a structured questionnaire (supplementary material 1). DNA Extractions and Polymerase Chain Reactions DNA extraction for Genotypes MTBDR sl was performed using Genolyse. Five microliters of the DNA were used for amplification, which was performed in an automated thermocycler (GeneAmp PCR system 9700). The PCR protocol was modified by increasing the number of cycles from 20 to 30 for the smear-positive samples and from 20 to 45 for the smear-negative samples (Hain Lifescience GmbH, Nehren, Germany, [ 16 ]). According to the Genotype MTBDRplus manufacturer’s instructions, the PCR product was detected by reverse hybridisation using Twincubator equipment (Hain Lifescience GmbH, Nehren, Germany). The results were interpreted and evaluated using the charts (Hain Lifescience GmbH, Nehren, Germany, [ 16 ]). Statistical analysis Categorical variables were expressed as frequencies. The chi-square test was used to determine the association between pre-XDR-TB and sociodemographic variables. Bivariate analyses were performed to examine factors associated with the frequency of pre-XDR-TB, using MedCalc Version 23.0.2 (Ostend, Belgium). All analyses with p -values < 0.05 at a 95% confidence interval (CI) were considered statistically significant. RESULTS Out of the 144 MDR-TB strains tested, the occurrence rate of pre-XDR was 24.3% (n=35) among 9.7% (35/360) individuals (Figure 1). Demographic factors associated with pre-XDR-TB infection among presumptive TB patients in Kaduna State are presented in Table 1. Patients aged ≤20 years had the highest frequency of pre-XDR-TB (14.29%), while the lowest frequency was observed in patients aged >60 years (5.71%). The frequency of pre-XDR-TB was relatively higher in males (11.88%) compared to females (8%). Based on the marital status of the patients, widows had the highest frequency of pre-XDR-TB (12.5%), while patients who were single (unmarried) had the lowest (9.13%). Patients without any formal education had the lowest frequency of pre-XDR-TB (6.72%), while patients with tertiary education had the highest frequency (15.39%). Based on patients' occupations, farmers had the lowest frequency of pre-MDR-TB (5.94%). There was no statistically significant association between the frequency of pre-MDR-TB and all sociodemographic variables ( p > 0.05) (Table 1). Analyses of risk factors associated with the frequency of pre-XDR-TB infection among patients with pulmonary illnesses in Kaduna State indicated that patients with low economic status (25%) had a relatively higher frequency of pre-XDR-TB compared to those with high economic status (9.38%) (Table 2). Patients with no history of previous TB infection (10.4%) had a relatively higher frequency of pre-XDR-TB. Based on HIV status, patients whose HIV status was unknown had a higher frequency of pre-XDR-TB (10.36%). Patients who do not smoke cigarettes (10.13%), consume alcohol (9.40%), and have no history of diabetes (09.84%) had a higher frequency of Pre-XDR-TB. Patients who had contact with TB patients had a relatively higher frequency of pre-XDR-TB (26.04%) than those who did not (3.76%). Patients who had previously received anti-tuberculosis treatment had a higher frequency of pre-XDR-TB (16.67%) compared to new case patients (7.8%). Of these, histories of contact with TB patients (OR=8.94, 95% CI: 4.1-19.49, p <0.0001) and prior anti-tuberculosis treatment (OR=2.36, 95% CI: 1.13-4.94, p =0.00233) were risk factors associated with pre-XDR-TB. (Table 2). Fluoroquinolone resistance was detected in 18/35 (51.4%) MDR-TB isolates due to diverse mutations and missing regions WT1 and WT2 in gyrA (n=14) (Table 3). Most chromosomal point mutations associated with FLQ resistance were detected in D94A, D94N/D94Y, D94G, and D94H of gyrA . The MTBDR sl assay identified mutations in the MUT1 (N538D) position of the QRDR gyrB among MDR-TB strains. The predominant mechanisms of aminoglycoside resistance (41.2%, 7/17) among pre-XDR-TB strains were MUT1 (A1401G) and MUT2 (G1484T) in the rrs gene. Moreover, the MUT1 (C-14T) mutation (23.5%, 4/17) was observed in the eis promoter region of the Second-Line Injectable Drugs-resistant isolates (Table 3). Furthermore, one isolate had a missing WT1 at the eis promoter region, which confers low-level resistance (Table 3). DISCUSSIONS Pre-XDR-TB has significant clinical consequences, including poorer treatment outcomes, higher risk of mortality and progression to XDR-TB. At the same time, public health consequences include faster spread, greater treatment burden, and the urgency of developing new drugs, necessitating robust surveillance for early detection, monitoring treatment progress, and informing targeted interventions. Nigeria faces a significant drug-resistant TB burden, with MDR-TB rates around 6% for new cases and 32% for previously treated cases [ 17 ]. Moreover, pre-XDR-TB is a growing concern; however, no study from northern Nigeria has identified the mechanisms underlying the phenotype, highlighting the need for improved molecular surveillance and diagnostics to optimise treatment strategies for patients' general well-being and to safeguard the general public. The 24.8% pre-XDR occurrence rate in MDR-TB in this study is significantly higher than in all previous data from Nigeria. For instance, 16.7% and 3.1% in southwest and southeastern Nigeria, respectively, as reported by Daniel et al. [ 18 ] and Nwachukwu et al. [ 19 ]. Although pre-XDR-TB has been reported in Nigeria over the past 20 years [ 18 ], to the best of our knowledge, no other locations have observed a similar shift in resistance from MDR to pre-XDR as ours. However, our pre-XDR-TB data corroborated the 26% (95% CI: 22–31%) from a global meta-analysis of pre-XDR in MDR-TB until 2022 [ 20 ]. Our findings highlight the ability of MDR strains to develop further resistance while effectively spreading without being identified in a lab, a significant limitation in traditional TB diagnostic methods used in most healthcare centres in Nigeria [ 17 ]. This is a significant public health concern because it leads to delayed or ineffective treatments, contributing to higher and unabated transmission in the community. We found that patients who had a history of contact with other TB patients had a significantly higher risk of developing pre-XDR-TB. This suggests that individuals in close contact (especially household contacts) with someone who has infectious pre-XDR-TB are at high risk of becoming infected with the same resistant strain [ 21 ]. The high frequency in this specific subgroup emphasises the critical need for systematic and timely screening and contact tracing for individuals exposed to drug-resistant TB cases. Additionally, our data revealed a strong correlation between the occurrence of pre-XDR-TB and prior antituberculosis history. This is comparable to the earlier research that showed a history of TB treatment cessation was a significant risk factor for pre-XDR-TB [ 22 ]. Furthermore, pre-XDR-TB has been linked to non-adherence to TB treatment in Nigeria [ 23 ]. About 12.5% and 11.2% of MDR-TB isolates were resistant to fluoroquinolone and aminoglycosides, respectively. Our data on second-line injectable AMGs were similar to 11% aminoglycoside-resistant MDR-TB in a global meta-analysis; however, that of the FLQ is lower than the 27% reported in the same study [ 20 ]. Nevertheless, it is essential to note that the 12.5% occurrence rate of FLQ-resistant MDR-TB is similar to the 12.88% previously reported in northwestern Nigeria during the COVID-19 pandemic [ 24 ] and 14.2% in southwestern Nigeria [ 25 ]. It appears that FLQ-resistant MDR-TB is relatively lower than the global data and those from individual studies from TB high-burden countries analysed by Diribi et al [ 20 ]. Findings from this study show that over 84% of FLQ-resistant isolates harboured a gyrA mutation, similar to previous studies in Nigeria and other Tb-endemic countries in Africa [ 25 , 26 , 27 , 28 ], indicating the predominance and persistence of this mechanism of resistance. In our study, polymorphisms in codons 91 and 94 of gyrA were identified, with mutations at codon 94 being the most prevalent. Previous data showed that codon 94 is the most commonly mutated codon, but the main mutation reported in this study was D94N/D94Y [ 29 ]. D94N/D94Y was the most prevalent mutation at codon 94 of gyrA . According to a systematic review, gyrA mutations in codons 85–94 appeared to account for roughly 60–90% of FLQ-resistant MDR-TB globally [ 26 ]. Moreover, three MDR-TB isolates from the present study harboured a mutation in gyrB (N538D), which is associated with low-level resistance to FLQ and occurs less frequently than the gyrA mutation. However, it’s widely recommended to consider the status of the gyrB gene when screening for pre-XDR-TB and XDR-TB isolates, especially since some FLQ-resistant strains lack gyrA mutations, thereby ensuring more accurate, rapid diagnosis and better treatment [ 12 , 30 ]. In our present study, over 72% of AMG-resistant MDR-TB had mutations in rrs gene, which confer high-level resistance to aminoglycosides, among which 44% were identified at codon A1401G mutation in rrs gene, encoding the 16S rRNA bacterial subunit, causing high-level resistance to kanamycin and cross-resistance to amikacin and seldom to capreomycin [ 31 ]. Aminoglycoside resistance in MDR-TB makes treatment much harder, leading to more extended, more toxic regimens, a higher risk of severe side effects like permanent deafness (ototoxicity), kidney damage (nephrotoxicity), and worse patient outcomes, potentially pushing towards XDR-TB with poorer prognosis and return to pre-antibiotic era risks, highlighting the need for better monitoring and new drug development. This study is not without limitations. The phylogenetic analysis of the pre-XDR-TB strains could have provided highly relevant data for public health and clinical management because it uses whole-genome sequencing data to track the transmission dynamics of the disease, identify the origins of outbreaks, and inform the development of targeted control strategies and individualised treatment plans. CONCLUSIONS This study revealed a high occurrence of pre-XDR-TB in the study population and among the MDR strains, which could lead to treatment failures and a higher public health threat. To stop pre-XDR-TB from spreading and growing and to improve treatment outcomes in this and other regions where it is more prevalent, it is imperative to diagnose resistance to second-line anti-tuberculosis quickly before beginning treatment and activate pre-XDR-TB surveillance systems. Abbreviations AMG: Aminoglycoside CI: Confidence Interval DST: Drug Susceptibility Testing FLQ: Fluoroquinolones HIV: Human Immunodeficiency Virus INH: Isoniazid MDR: MultiDrug Resistant MTB: Mycobacterium tuberculosis MUT: Mutation NHREC: Nigeria Human Research Ethics Committee NTBLTC: National Tuberculosis and Leprosy Training Centre OR: Odd Ratio p : probability PCR: Polymerase Chain Reaction Pre-XDR-TB: Pre-extensively drug-resistant Tuberculosis RIF: Rifampicin RIPE: Rifampin, Isoniazid, Pyrazinamide, Ethambutol WT: Wild Type XDR: Extensively-Drug-Resistant Declarations Consent for publication Not applicable Approval declaration This study was approved by the ethical research committee of the National Tuberculosis and Leprosy Training Centre (NTBLTC and the Research Ethics Committee of Kaduna State Ministry of Health, Nigeria (approved number: NHREC/17/03/2018). Consent to participate Written informed consent was obtained from all the participants and from the legal guardians of the participants who were illiterate Human Ethics This study was performed in accordance with the Declaration of Helsinki. Clinical trial number Not applicable. Availability of data and materials The tables, figures, and supplementary materials presented all the data generated from this study. However, further requests could be made through the corresponding author. Competing interests None declared by the authors. Funding None received Acknowledgements Not applicable Funding Declaration None received Authors’ contributions K.M.: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. M.S.A.: Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Conceptualization. M.S.: Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. A.K.I.: Writing – review & editing, Writing – original draft, Validation, Resources, Methodology, Investigation, Data curation, Conceptualization. A.M.S.: Writing – review & editing, Writing – original draft, Validation, Resources, Methodology, Investigation, Formal analysis, Data curation. B.Y.G: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. D.A.A.: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. M.Z.D.: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. G.M.: Writing – review & editing, Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis. H.M.S.: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation. I.M.H.: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation. I.N.A.: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. References Suleac M, Rezende A, Naranjo S, Djassi M. A Case of Extensive Tuberculosis With Bacterial Infection Treated in a Peripheral Hospital. Cureus. 2024;16(3):e57067. 10.7759/cureus.57067 . PMID: 38681350; PMCID: PMC11052601. 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Evaluation of the GenoType MTBDR sl Version 2.0 Assay for Second-Line Drug Resistance Detection of Mycobacterium tuberculosis Isolates in South Africa. J Clin Microbiol. 2017;55(3):791–800. Epub 2016 Dec 14. PMID: 27974543; PMCID: PMC5328447. Feuerriegel S, Oberhauser B, George AG, Dafae F, Richter E, Rüsch-Gerdes S, Niemann S. Sequence analysis for detection of first-line drug resistance in Mycobacterium tuberculosis strains from a high-incidence setting. BMC Microbiol. 2012;12:90. 10.1186/1471-2180-12-90 . PMID: 22646308; PMCID: PMC3404943. Bablishvili N, Tukvadze N, Shashkina E, Mathema B, Gandhi NR, Blumberg HM, Kempker RR. Impact of gyrB and eis Mutations in Improving Detection of Second-Line-Drug Resistance among Mycobacterium tuberculosis Isolates from Georgia. Antimicrob Agents Chemother. 2017;61(9):e01921–16. 10.1128/AAC.01921-16 . PMID: 28630205; PMCID: PMC5571295. Hu Y, Hoffner S, Wu L, Zhao Q, Jiang W, Xu B. Prevalence and genetic characterization of second-line drug-resistant and extensively drug-resistant Mycobacterium tuberculosis in Rural China. Antimicrob Agents Chemother. 2013;57(8):3857–63. 10.1128/AAC.00102-13 . Epub 2013 Jun 3. PMID: 23733477; PMCID: PMC3719720. Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files MTBSupplmaterial1.pdf Tables.docx Cite Share Download PDF Status: Published Journal Publication published 28 Mar, 2026 Read the published version in BMC Infectious Diseases → Version 1 posted Editorial decision: Revision requested 05 Mar, 2026 Reviews received at journal 27 Feb, 2026 Reviews received at journal 25 Feb, 2026 Reviews received at journal 20 Feb, 2026 Reviewers agreed at journal 20 Feb, 2026 Reviews received at journal 18 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers agreed at journal 17 Feb, 2026 Reviewers agreed at journal 14 Feb, 2026 Reviewers agreed at journal 11 Feb, 2026 Reviewers invited by journal 08 Feb, 2026 Editor assigned by journal 06 Feb, 2026 Editor invited by journal 16 Jan, 2026 Submission checks completed at journal 16 Jan, 2026 First submitted to journal 16 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Sani","lastName":"Aliyu","suffix":""},{"id":589384158,"identity":"dbf75bc5-4430-439a-81d3-9e7918735608","order_by":2,"name":"Mikailu Suleman","email":"","orcid":"","institution":"National Institute of Public Health and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Mikailu","middleName":"","lastName":"Suleman","suffix":""},{"id":589384159,"identity":"8ebd84ee-b655-4253-a0c6-e16859046a76","order_by":3,"name":"Abba Kasim Ibrahim","email":"","orcid":"","institution":"National Institute of Public Health and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Abba","middleName":"Kasim","lastName":"Ibrahim","suffix":""},{"id":589384160,"identity":"f305a756-221c-4535-92d3-515956356273","order_by":4,"name":"Abubakar Mohammed Song","email":"","orcid":"","institution":"National Institute of Public Health and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Abubakar","middleName":"Mohammed","lastName":"Song","suffix":""},{"id":589384161,"identity":"657a7a6c-9a17-4002-8c59-1683ad846f09","order_by":5,"name":"Bala Yazeed Garba","email":"","orcid":"","institution":"National Institute of Public Health and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Bala","middleName":"Yazeed","lastName":"Garba","suffix":""},{"id":589384162,"identity":"1170ad5f-fae6-417d-8338-373c111d3101","order_by":6,"name":"Dalhatu Abdullahi Aminu","email":"","orcid":"","institution":"National Institute of Public Health and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Dalhatu","middleName":"Abdullahi","lastName":"Aminu","suffix":""},{"id":589384168,"identity":"11a79714-a694-4a71-ba18-734144411260","order_by":7,"name":"Muhammad Zaharadeen Dan-Inna","email":"","orcid":"","institution":"Federal Medical Center Gusau","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Zaharadeen","lastName":"Dan-Inna","suffix":""},{"id":589384170,"identity":"31e61bc0-73a1-49e8-bd15-ebc57ea514cb","order_by":8,"name":"Gaius Mathew","email":"","orcid":"","institution":"Department of Medical Laboratory Services, Federal Ministry of Health, Abuja","correspondingAuthor":false,"prefix":"","firstName":"Gaius","middleName":"","lastName":"Mathew","suffix":""},{"id":589384171,"identity":"7979c950-d694-483c-a60d-61250ef93b99","order_by":9,"name":"Hamisu Muhammed Salihu","email":"","orcid":"","institution":"Kano Independent Research Centre Trust","correspondingAuthor":false,"prefix":"","firstName":"Hamisu","middleName":"Muhammed","lastName":"Salihu","suffix":""},{"id":589384173,"identity":"c2a6cf4f-7003-4e8a-af52-4d1a1ffc918b","order_by":10,"name":"Ibrahim Mohammed Hussaini","email":"","orcid":"","institution":"Ahmadu Bello University","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"Mohammed","lastName":"Hussaini","suffix":""},{"id":589384175,"identity":"8e46a12b-545a-40d6-8219-d5e2651bc281","order_by":11,"name":"Idris Nasir Abdullahi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBACNhDiYbCQYZBgbHzA2AASSyBKiwQQMTYbEKWFAaGFgU2CKC180s3PHrxtk+Dhn93cVvFzx2EGfvYcA+aCX3iskDlmbjgXqEXizsG2m71nDjNI9rwxYJ7Zh0eLRIKZNC9QC8ONxLYbvG2HGQxuAG3h7cGnJf0bWIs8UEvhX6AWe8JaciC2GAC1MINtkQBq4fmBV0u54ZxzEjyGNxKbpWXPpPNInHlWcJi3AbcW+Rnp2x68KbORk7uR/vDj2x3WcvztyRsf8/zBrQUD8ICIA4xtJGiBAlJsGQWjYBSMguEOAEQ3TE8TBkvSAAAAAElFTkSuQmCC","orcid":"","institution":"Ahmadu Bello University","correspondingAuthor":true,"prefix":"","firstName":"Idris","middleName":"Nasir","lastName":"Abdullahi","suffix":""}],"badges":[],"createdAt":"2026-01-07 09:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8539120/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8539120/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12879-026-13191-z","type":"published","date":"2026-03-28T16:13:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":102596102,"identity":"49f87569-b66d-49a6-b864-d80c36c53142","added_by":"auto","created_at":"2026-02-13 12:14:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55563,"visible":true,"origin":"","legend":"\u003cp\u003ePattern of drug resistance among MDR-TB strains from patients with pulmonary illnesses in Kaduna State, Nigeria.\u003c/p\u003e\n\u003cp\u003eKey: AMG: Aminoglycosides, FLQ: Fluoroquinolones, MDR: Multidrug-resistant, pre-XDR: pre-extensively drug-resistant, TB: Tuberculosis\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8539120/v1/f20d21980bfa9098344904eb.png"},{"id":105756070,"identity":"11047ab4-0b60-4d6c-b974-fabb0d08cf40","added_by":"auto","created_at":"2026-03-30 16:35:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":746059,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8539120/v1/2234a215-ba2a-4feb-8c25-8386018275b2.pdf"},{"id":102747292,"identity":"4a82f046-30d8-44fb-86d4-4796959a1c11","added_by":"auto","created_at":"2026-02-16 09:04:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":111477,"visible":true,"origin":"","legend":"","description":"","filename":"MTBSupplmaterial1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8539120/v1/48ffec38870ca1d0d8481579.pdf"},{"id":102596103,"identity":"fc6f306c-9432-4d29-8b21-420e9c3ecb7a","added_by":"auto","created_at":"2026-02-13 12:14:37","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":34907,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8539120/v1/e3d073158289c802265a690f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Frequency, genetic mechanisms and factors associated with pre-extensively drug-resistant tuberculosis in Northern Nigeria","fulltext":[{"header":"INTRODUCTIONS","content":"\u003cp\u003eRifampicin-resistant \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e (MTB), often referred to as multidrug-resistant TB (MDR-TB), is a critical priority pathogen and one of the leading 10 causes of infection-associated death, especially in developing countries [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nigeria is the top in Africa and ranked 6th among the high TB-burdened countries in the world [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], contributing to 4.5% of the estimated global incidence [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTreating TB is crucial to save lives from this leading infectious killer, prevent severe illness and disability, stop its airborne spread, and combat the dangerous rise of drug-resistant strains by ensuring the bacteria are eliminated, preventing relapse, and protecting the wider community. The standard first-line treatment for drug-susceptible TB involves a two-phase regimen using a combination of four core antibiotics, often remembered by the acronym RIPE (Rifampin, Isoniazid, Pyrazinamide, Ethambutol) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The need for treating TB with second-line drugs arises when the standard, or first-line, medications are not effective due to drug resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While essential, second-line drugs often require a longer course of treatment (up to two years), can be more toxic, and are associated with frequent adverse events compared to first-line regimens. Therefore, treatment decisions are guided by drug susceptibility testing (DST) to ensure the most effective regimen is selected for each patient, tailored to their specific resistance profile [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMDR occurs when MTB is resistant to rifampicin (RIF) and isoniazid (INH) with or without other first-line drugs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In 2023, a global meta-analysis reported that 9% of MDR-TB patients had developed extremely drug-resistant TB (XDR-TB) strains, while 26% had pre-XDR-TB [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. XDR-TB is a severe, rare form of TB where the bacterium is resistant to first-line drugs (isoniazid, rifampin) and also to a fluoroquinolone and at least one second-line injectable drug, making it extremely difficult to treat with standard options, leading to longer, more toxic treatments and a higher risk of severe outcomes, especially for people with weakened immune systems such as those caused by human immunodeficiency virus (HIV) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eXDR-TB is defined as MDR-TB that is resistant to any fluoroquinolone and at least one of the three aminoglycoside drugs. Conversely, pre-XDR-TB is defined when the bacterium is resistant to either (a) Isoniazid, rifampin, and a fluoroquinolone or (b) Isoniazid, rifampin, and a second-line injectable (amikacin, capreomycin, and kanamycin) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFluoroquinolones (FLQs) and aminoglycosides (AMGs) have long been used as anti-tuberculosis drugs, and their widespread use has led to the development of resistance in clinical isolates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. FLQ resistance in TB is primarily driven by mutations in the quinolone resistance-determining region (QRDR) of \u003cem\u003egyrA\u003c/em\u003e (subunit A) and, less commonly, \u003cem\u003egyrB\u003c/em\u003e (subunit B), which encode DNA gyrase, the enzyme targeted by these essential second-line anti-TB drugs, with \u003cem\u003egyrA\u003c/em\u003e mutations (especially at codons 88\u0026ndash;94) being most frequent and conferring higher resistance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMutations in the \u003cem\u003errs\u003c/em\u003e and overexpression of the \u003cem\u003eeis\u003c/em\u003e genes are the primary mechanisms responsible for aminoglycoside resistance in MTB [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The \u003cem\u003errs\u003c/em\u003e gene mutations in the 16S ribosomal RNA gene alter the drug's ribosomal target site, preventing effective binding and conferring high-level resistance to amikacin and kanamycin and moderate resistance to capreomycin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The A1401G mutation is the most frequent mutation associated with high-level resistance to amikacin and kanamycin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Mutations or overexpression of the \u003cem\u003eeis\u003c/em\u003e gene promoter increase expression of the \u003cem\u003eeis\u003c/em\u003e gene, producing more of the Eis enzyme. This enzyme inactivates aminoglycosides by acetylating them (adding an acetyl group), preventing the drug from inhibiting protein synthesis. This mechanism typically confers a low-level resistance to kanamycin but does not usually cause clinically significant resistance to amikacin [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo prioritise and facilitate the identification of pre-XDR-TB and XDR-TB, several molecular testing platforms, such as GenoType MTBDRsl VER 2.0, have improved the detection and management of drug-resistant TB [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This study aims to determine the frequency and mutations associated with fluoroquinolone and aminoglycoside resistance in MTB isolates from patients with pulmonary illnesses in Kaduna State, Nigeria.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area and population\u003c/h2\u003e \u003cp\u003eOne hundred and forty-four (144) frozen sputum samples containing MDR-TB previously collected from people with pulmonary illnesses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] were thawed and re-analysed at the National Tuberculosis and Leprosy Training Centre (NTLTC), Zaria, Nigeria. The samples were decontaminated using NaOH-Na-Citrate-NALC method. An inoculum of 0.1mL of decontaminated sputum sample was prepared and incubated at 37\u0026deg;C for 8 weeks. Colonies from the positive culture slants were used to identify the MTB complex using rapid immunochromatographic test kits.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy design and data collection\u003c/h3\u003e\n\u003cp\u003eThis hospital-based surveillance study included individuals seeking healthcare at NTLTC, regardless of age or sex. Data from this study were collected using a structured questionnaire \u003cb\u003e(supplementary material 1).\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003eDNA Extractions and Polymerase Chain Reactions\u003c/h3\u003e\n\u003cp\u003eDNA extraction for Genotypes MTBDR\u003cem\u003esl\u003c/em\u003e was performed using Genolyse. Five microliters of the DNA were used for amplification, which was performed in an automated thermocycler (GeneAmp PCR system 9700). The PCR protocol was modified by increasing the number of cycles from 20 to 30 for the smear-positive samples and from 20 to 45 for the smear-negative samples (Hain Lifescience GmbH, Nehren, Germany, [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]). According to the Genotype MTBDRplus manufacturer\u0026rsquo;s instructions, the PCR product was detected by reverse hybridisation using Twincubator equipment (Hain Lifescience GmbH, Nehren, Germany). The results were interpreted and evaluated using the charts (Hain Lifescience GmbH, Nehren, Germany, [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eCategorical variables were expressed as frequencies. The chi-square test was used to determine the association between pre-XDR-TB and sociodemographic variables. Bivariate analyses were performed to examine factors associated with the frequency of pre-XDR-TB, using MedCalc Version 23.0.2 (Ostend, Belgium). All analyses with \u003cem\u003ep\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 at a 95% confidence interval (CI) were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eOut of the 144 MDR-TB strains tested, the occurrence rate of pre-XDR was 24.3% (n=35) among 9.7% (35/360) individuals \u003cstrong\u003e(Figure 1).\u003c/strong\u003e\u0026nbsp; Demographic factors associated with pre-XDR-TB infection among presumptive TB patients in Kaduna State are presented in \u003cstrong\u003eTable 1.\u003c/strong\u003e Patients aged ≤20 years had the highest frequency of pre-XDR-TB (14.29%), while the lowest frequency was observed in patients aged \u0026gt;60 years (5.71%). The frequency of pre-XDR-TB was relatively higher in males (11.88%) compared to females (8%). Based on the marital status of the patients, widows had the highest frequency of pre-XDR-TB (12.5%), while patients who were single (unmarried) had the lowest (9.13%). Patients without any formal education had the lowest frequency of pre-XDR-TB (6.72%), while patients with tertiary education had the highest frequency (15.39%). Based on patients' occupations, farmers had the lowest frequency of pre-MDR-TB (5.94%). There was no statistically significant association between the frequency of pre-MDR-TB and all sociodemographic variables (\u003cem\u003ep\u003c/em\u003e\u0026gt; 0.05) \u003cstrong\u003e(Table 1).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalyses of risk factors associated with the frequency of pre-XDR-TB infection among patients with pulmonary illnesses in Kaduna State indicated that patients with low economic status (25%) had a relatively higher frequency of pre-XDR-TB compared to those with high economic status (9.38%) \u003cstrong\u003e(Table 2).\u003c/strong\u003e Patients with no history of previous TB infection (10.4%) had a relatively higher frequency of pre-XDR-TB. Based on HIV status, patients whose HIV status was unknown had a higher frequency of pre-XDR-TB (10.36%). Patients who do not smoke cigarettes (10.13%), consume alcohol (9.40%), and have no history of diabetes (09.84%) had a higher frequency of Pre-XDR-TB. Patients who had contact with TB patients had a relatively higher frequency of pre-XDR-TB (26.04%) than those who did not (3.76%). Patients who had previously received anti-tuberculosis treatment had a higher frequency of pre-XDR-TB (16.67%) compared to new case patients (7.8%). Of these, histories of contact with TB patients (OR=8.94, 95% CI: 4.1-19.49, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001) and prior anti-tuberculosis treatment (OR=2.36, 95% CI: 1.13-4.94, \u003cem\u003ep\u003c/em\u003e=0.00233) were risk factors associated with pre-XDR-TB. \u003cstrong\u003e(Table 2).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluoroquinolone resistance was detected in 18/35 (51.4%) MDR-TB isolates due to diverse mutations and missing regions WT1 and WT2 in \u003cem\u003egyrA\u003c/em\u003e (n=14) \u003cstrong\u003e(Table 3).\u003c/strong\u003e Most chromosomal point mutations associated with FLQ resistance were detected in D94A, D94N/D94Y, D94G, and D94H of \u003cem\u003egyrA\u003c/em\u003e. The MTBDR\u003cem\u003esl\u0026nbsp;\u003c/em\u003eassay identified mutations in the MUT1 (N538D) position of the QRDR \u003cem\u003egyrB\u003c/em\u003e among MDR-TB strains. The predominant mechanisms of aminoglycoside resistance (41.2%, 7/17) among pre-XDR-TB strains were MUT1 (A1401G) and MUT2 (G1484T) in the \u003cem\u003errs\u0026nbsp;\u003c/em\u003egene. Moreover, the MUT1 (C-14T) mutation (23.5%, 4/17) was observed in the \u003cem\u003eeis\u003c/em\u003e promoter region of the Second-Line Injectable Drugs-resistant isolates (Table 3). Furthermore, one isolate had a missing WT1 at the \u003cem\u003eeis\u003c/em\u003e promoter region, which confers low-level resistance \u003cstrong\u003e(Table 3).\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSIONS","content":"\u003cp\u003ePre-XDR-TB has significant clinical consequences, including poorer treatment outcomes, higher risk of mortality and progression to XDR-TB. At the same time, public health consequences include faster spread, greater treatment burden, and the urgency of developing new drugs, necessitating robust surveillance for early detection, monitoring treatment progress, and informing targeted interventions.\u003c/p\u003e \u003cp\u003eNigeria faces a significant drug-resistant TB burden, with MDR-TB rates around 6% for new cases and 32% for previously treated cases [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, pre-XDR-TB is a growing concern; however, no study from northern Nigeria has identified the mechanisms underlying the phenotype, highlighting the need for improved molecular surveillance and diagnostics to optimise treatment strategies for patients' general well-being and to safeguard the general public.\u003c/p\u003e \u003cp\u003eThe 24.8% pre-XDR occurrence rate in MDR-TB in this study is significantly higher than in all previous data from Nigeria. For instance, 16.7% and 3.1% in southwest and southeastern Nigeria, respectively, as reported by Daniel et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and Nwachukwu et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although pre-XDR-TB has been reported in Nigeria over the past 20 years [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], to the best of our knowledge, no other locations have observed a similar shift in resistance from MDR to pre-XDR as ours. However, our pre-XDR-TB data corroborated the 26% (95% CI: 22\u0026ndash;31%) from a global meta-analysis of pre-XDR in MDR-TB until 2022 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Our findings highlight the ability of MDR strains to develop further resistance while effectively spreading without being identified in a lab, a significant limitation in traditional TB diagnostic methods used in most healthcare centres in Nigeria [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This is a significant public health concern because it leads to delayed or ineffective treatments, contributing to higher and unabated transmission in the community.\u003c/p\u003e \u003cp\u003eWe found that patients who had a history of contact with other TB patients had a significantly higher risk of developing pre-XDR-TB. This suggests that individuals in close contact (especially household contacts) with someone who has infectious pre-XDR-TB are at high risk of becoming infected with the same resistant strain [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The high frequency in this specific subgroup emphasises the critical need for systematic and timely screening and contact tracing for individuals exposed to drug-resistant TB cases.\u003c/p\u003e \u003cp\u003eAdditionally, our data revealed a strong correlation between the occurrence of pre-XDR-TB and prior antituberculosis history. This is comparable to the earlier research that showed a history of TB treatment cessation was a significant risk factor for pre-XDR-TB [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, pre-XDR-TB has been linked to non-adherence to TB treatment in Nigeria [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAbout 12.5% and 11.2% of MDR-TB isolates were resistant to fluoroquinolone and aminoglycosides, respectively. Our data on second-line injectable AMGs were similar to 11% aminoglycoside-resistant MDR-TB in a global meta-analysis; however, that of the FLQ is lower than the 27% reported in the same study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Nevertheless, it is essential to note that the 12.5% occurrence rate of FLQ-resistant MDR-TB is similar to the 12.88% previously reported in northwestern Nigeria during the COVID-19 pandemic [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and 14.2% in southwestern Nigeria [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It appears that FLQ-resistant MDR-TB is relatively lower than the global data and those from individual studies from TB high-burden countries analysed by Diribi et al [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFindings from this study show that over 84% of FLQ-resistant isolates harboured a \u003cem\u003egyrA\u003c/em\u003e mutation, similar to previous studies in Nigeria and other Tb-endemic countries in Africa [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], indicating the predominance and persistence of this mechanism of resistance. In our study, polymorphisms in codons 91 and 94 of \u003cem\u003egyrA\u003c/em\u003e were identified, with mutations at codon 94 being the most prevalent. Previous data showed that codon 94 is the most commonly mutated codon, but the main mutation reported in this study was D94N/D94Y [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. D94N/D94Y was the most prevalent mutation at codon 94 of \u003cem\u003egyrA\u003c/em\u003e. According to a systematic review, \u003cem\u003egyrA\u003c/em\u003e mutations in codons 85\u0026ndash;94 appeared to account for roughly 60\u0026ndash;90% of FLQ-resistant MDR-TB globally [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Moreover, three MDR-TB isolates from the present study harboured a mutation in \u003cem\u003egyrB\u003c/em\u003e (N538D), which is associated with low-level resistance to FLQ and occurs less frequently than the \u003cem\u003egyrA\u003c/em\u003e mutation. However, it\u0026rsquo;s widely recommended to consider the status of the \u003cem\u003egyrB\u003c/em\u003e gene when screening for pre-XDR-TB and XDR-TB isolates, especially since some FLQ-resistant strains lack \u003cem\u003egyrA\u003c/em\u003e mutations, thereby ensuring more accurate, rapid diagnosis and better treatment [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our present study, over 72% of AMG-resistant MDR-TB had mutations in \u003cem\u003errs\u003c/em\u003e gene, which confer high-level resistance to aminoglycosides, among which 44% were identified at codon A1401G mutation in \u003cem\u003errs\u003c/em\u003e gene, encoding the 16S rRNA bacterial subunit, causing high-level resistance to kanamycin and cross-resistance to amikacin and seldom to capreomycin [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Aminoglycoside resistance in MDR-TB makes treatment much harder, leading to more extended, more toxic regimens, a higher risk of severe side effects like permanent deafness (ototoxicity), kidney damage (nephrotoxicity), and worse patient outcomes, potentially pushing towards XDR-TB with poorer prognosis and return to pre-antibiotic era risks, highlighting the need for better monitoring and new drug development.\u003c/p\u003e \u003cp\u003eThis study is not without limitations. The phylogenetic analysis of the pre-XDR-TB strains could have provided highly relevant data for public health and clinical management because it uses whole-genome sequencing data to track the transmission dynamics of the disease, identify the origins of outbreaks, and inform the development of targeted control strategies and individualised treatment plans.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis study revealed a high occurrence of pre-XDR-TB in the study population and among the MDR strains, which could lead to treatment failures and a higher public health threat. To stop pre-XDR-TB from spreading and growing and to improve treatment outcomes in this and other regions where it is more prevalent, it is imperative to diagnose resistance to second-line anti-tuberculosis quickly before beginning treatment and activate pre-XDR-TB surveillance systems.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAMG: Aminoglycoside\u003c/p\u003e\n\u003cp\u003eCI: Confidence Interval\u003c/p\u003e\n\u003cp\u003eDST: Drug Susceptibility Testing\u003c/p\u003e\n\u003cp\u003eFLQ: Fluoroquinolones\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHIV: Human Immunodeficiency Virus\u003c/p\u003e\n\u003cp\u003eINH: Isoniazid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMDR: MultiDrug Resistant\u003c/p\u003e\n\u003cp\u003eMTB:\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMUT: Mutation\u003c/p\u003e\n\u003cp\u003eNHREC: Nigeria Human Research Ethics Committee\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNTBLTC:\u0026nbsp;National Tuberculosis and Leprosy Training Centre\u003c/p\u003e\n\u003cp\u003eOR: Odd Ratio\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e: probability\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003ePre-XDR-TB: Pre-extensively drug-resistant\u0026nbsp;Tuberculosis\u003c/p\u003e\n\u003cp\u003eRIF: Rifampicin\u003c/p\u003e\n\u003cp\u003eRIPE: Rifampin, Isoniazid, Pyrazinamide, Ethambutol\u003c/p\u003e\n\u003cp\u003eWT: Wild Type\u003c/p\u003e\n\u003cp\u003eXDR: Extensively-Drug-Resistant\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApproval declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the ethical research committee of\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethe National Tuberculosis and Leprosy Training Centre (NTBLTC and the Research Ethics Committee of Kaduna State Ministry of Health, Nigeria (approved number: NHREC/17/03/2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all the participants and from the legal guardians of the participants who were illiterate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in accordance with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tables, figures, and supplementary materials presented all the data generated from this study. However, further requests could be made through the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone declared by the authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone received\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone received\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.M.: Writing – review \u0026amp; editing, Writing – original draft, Visualization, Validation, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. M.S.A.: Writing – review \u0026amp; editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Conceptualization. M.S.: Writing – review \u0026amp; editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. A.K.I.: Writing – review \u0026amp; editing, Writing – original draft, Validation, Resources, Methodology, Investigation, Data curation, Conceptualization. A.M.S.: Writing – review \u0026amp; editing, Writing – original draft, Validation, Resources, Methodology, Investigation, Formal analysis, Data curation. B.Y.G: Writing – review \u0026amp; editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. D.A.A.: Writing – review \u0026amp; editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. M.Z.D.: Writing – review \u0026amp; editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. G.M.: Writing – review \u0026amp; editing, Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis. H.M.S.: Writing – review \u0026amp; editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation. I.M.H.: Writing – review \u0026amp; editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation. I.N.A.: Writing – review \u0026amp; editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSuleac M, Rezende A, Naranjo S, Djassi M. A Case of Extensive Tuberculosis With Bacterial Infection Treated in a Peripheral Hospital. 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Antimicrob Agents Chemother. 2013;57(8):3857\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/AAC.00102-13\u003c/span\u003e\u003cspan address=\"10.1128/AAC.00102-13\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 2013 Jun 3. PMID: 23733477; PMCID: PMC3719720.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tuberculosis, rrs, eis, pre-XDR-TB, Drug-resistant TB, Nigeria","lastPublishedDoi":"10.21203/rs.3.rs-8539120/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8539120/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRifampicin-resistant \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e (MTB), a critical priority pathogen, becomes highly complicated when it develops into pre-extensively drug-resistant (pre-XDR) and is an exceptional challenge for global TB control efforts. This study aims to determine the frequency and mutations associated with fluoroquinolone and aminoglycoside resistance in MTB isolates from patients with pulmonary illnesses in Kaduna State, Nigeria.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eA total of 144 MDR-TB-positive isolates previously obtained from sputum samples were collected from 360 individuals and were processed using the NaOH-Na-citrate-NALC method. All isolates were tested for MTB complex using TB Ag MPT64 (SD Bioline), and GenoType MTBDR\u003cem\u003esl\u003c/em\u003e VER 2.0 was used to identify chromosomal mutations in \u003cem\u003egyrA, gyrB, rrs\u003c/em\u003e and \u003cem\u003eeis\u003c/em\u003e genes. Risk associated with pre-XDR-TB was assessed using a structured questionnaire, and the generated data were statistically analysed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe occurrence rate of pre-XDR in MDR-TB was 24.3% (n\u0026thinsp;=\u0026thinsp;35) among 9.7% of individuals. Fluoroquinolone resistance was detected in 18/35 (51.4%) due to diverse mutations and missing regions WT1 and WT2 in \u003cem\u003egyrA\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;14) and N538D in \u003cem\u003egyrB\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4). The predominant mechanisms of aminoglycoside resistance (41.2%, 7/17) among pre-XDR-TB strains were MUT1 (A1401G) and MUT2 (G1484T) in the \u003cem\u003errs\u003c/em\u003e gene. Moreover, the MUT1 (C-14T) mutation (23.5%, 4/17) was observed in the \u003cem\u003eeis\u003c/em\u003e promoter region. Histories of contact with TB patients (OR\u0026thinsp;=\u0026thinsp;8.94, 95% CI: 4.1-19.49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and prior anti-tuberculosis treatment (OR\u0026thinsp;=\u0026thinsp;2.36, 95% CI: 1.13\u0026ndash;4.94, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00233) were associated with pre-XDR-TB.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study revealed a high occurrence of pre-XDR-TB in the study population and among the MDR strains, which could lead to treatment failures and a higher public health threat. To stop pre-XDR-TB from spreading and growing and to improve treatment outcomes in this and other regions where it is more prevalent, it is imperative to diagnose resistance to second-line anti-tuberculosis quickly before beginning treatment and activate pre-XDR-TB surveillance systems.\u003c/p\u003e","manuscriptTitle":"Frequency, genetic mechanisms and factors associated with pre-extensively drug-resistant tuberculosis in Northern Nigeria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 12:14:32","doi":"10.21203/rs.3.rs-8539120/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-05T06:38:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-27T13:26:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T10:26:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-20T12:35:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21853359095430205557323518152610910088","date":"2026-02-20T10:22:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-18T18:07:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139126883572806416675868607654779254037","date":"2026-02-18T09:22:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91293567667090271893601728942533006044","date":"2026-02-17T10:18:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50352212380661146645346490871684258067","date":"2026-02-14T09:34:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106671953629538421281633062986280914542","date":"2026-02-11T06:01:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-09T04:20:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-06T07:39:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-16T09:02:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-16T06:41:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2026-01-16T06:35:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"95a869d0-85e2-4cb5-9635-b15e8ed43a61","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:31:33+00:00","versionOfRecord":{"articleIdentity":"rs-8539120","link":"https://doi.org/10.1186/s12879-026-13191-z","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2026-03-28 16:13:01","publishedOnDateReadable":"March 28th, 2026"},"versionCreatedAt":"2026-02-13 12:14:32","video":"","vorDoi":"10.1186/s12879-026-13191-z","vorDoiUrl":"https://doi.org/10.1186/s12879-026-13191-z","workflowStages":[]},"version":"v1","identity":"rs-8539120","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8539120","identity":"rs-8539120","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}