Tuberculosis (TB) remains a major global public health problem, particularly in low-resource countries, where it is often aggravated by co-infection with the human immunodeficiency virus (HIV). HIV-induced immunosuppression worsens the clinical prognosis and complicates therapeutic management [1].

Despite progress achieved in diagnosis and treatment, the increasing incidence of drug-resistant tuberculosis seriously compromises treatment success. In a worrying evolving dynamic, inadequate patient management, treatment interruption, direct transmission of resistant strains, or spontaneous genetic mutations in mycobacteria (particularly in the katG and rpoB genes) may lead to a worsening resistance profile [2]. Consequently, increasingly complex resistant strains of Mycobacterium tuberculosis, particularly multidrug-resistant tuberculosis (MDR-TB), are emerging worldwide [3].

MDR-TB is defined as an infection caused by a mycobacterium belonging to the Mycobacterium tuberculosis complex that is resistant to at least isoniazid and rifampicin, the two most powerful first-line anti-tuberculosis drugs [2][4]. In 2018, the World Health Organization (WHO) estimated that approximately half a million new cases of rifampicin-resistant TB occurred worldwide, of which 78% were MDR-TB cases, highlighting rifampicin resistance as a poor prognostic factor [4][5].

According to the WHO in 2024, approximately 57,000 people in the African region developed multidrug-resistant or rifampicin-resistant tuberculosis, representing 15% of the global burden [6]. Chad is not spared from this burden, where the rate of drug-resistant tuberculosis in the general population remains significant [7]. The incidence of multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB) is estimated at 2.4 cases per 100,000 inhabitants, corresponding to approximately 420 estimated MDR-TB cases. The proportion of MDR-TB is estimated at 1.5% among new cases and 3.7% among previously treated cases [8]. According to the National Tuberculosis Control Program (PNT), the number of laboratory-confirmed drug-resistant TB cases does not reach half of the expected cases [9].

Among people living with HIV (PLHIV), the management of MDR-TB is even more complex due to the combined challenges associated with immunosuppression. Early identification of MDR-TB cases among PLHIV, adaptation of therapeutic strategies, and control of disease transmission remain constant concerns for clinicians [4][5].

However, despite the strong interaction between HIV and tuberculosis, specific data on multidrug resistance among HIV/TB co-infected patients remain limited. Several studies have highlighted that fewer than half of sub-Saharan African countries have reliable data on multidrug-resistant TB, reflecting a substantial gap in surveillance and research [10].

In the Chadian context, where the dual burden of tuberculosis and HIV remains high [11][12], epidemiological data on multidrug-resistant tuberculosis among HIV/TB co-infected patients are particularly scarce or even nonexistent at the national level, despite the National Tuberculosis Control Program’s recommendation to use Xpert MTB/RIF in all sites equipped with GeneXpert devices [13][9]. This gap constitutes a major obstacle to the development of appropriate management strategies. Therefore, the present study aimed to detect the mutations in genes associated with multidrug resistance to anti-tuberculosis drugs in the Mycobacterium tuberculosis complex among HIV/TB co-infected patients in N’Djamena, Chad.

In this study, the presence of gene mutations in Mycobacterium tuberculosis associated with resistance to various anti-tuberculosis drugs among HIV/TB co-infected patients was investigated using the Xpert MTB/RIF and Xpert MTB/XDR assays.

Mutations in the rpoB gene responsible for rifampicin resistance were detected in 4 patients, corresponding to a rate of 4.12% (4/97). This result is comparable to those reported in South Africa by Nazir et al. (4.9% in 2012) [19], in Chad by Ossoga et al. (5.21% in 2012) [7], and in Nigeria by Nwalozie et al. and Ibrahim et al. (5.42% and 4.2% in 2015 and 2018, respectively) [20][21]. However, other African studies have reported higher proportions than in the present study, such as Ogumbo et al. in Kenya (6.7% in 2022) [22] and Atemie et al. in Nigeria (9.4% in 2024) [23]. These geographical variations may be influenced by differences in access to rapid molecular diagnostics (such as GeneXpert MTB/RIF), access to medications, treatment adherence, and previous anti-tuberculosis treatment history. The presence of rifampicin resistance among co-infected patients is clinically significant, as it complicates management, increases the risk of treatment failure, and may contribute to the transmission of resistant strains in vulnerable populations.

Regarding isoniazid resistance, mutations were detected in 9 patients, corresponding to a rate of 9.27%. This rate is broadly comparable to findings reported in Nigeria by Lana et al. in 2012 (9.3%) [24] and in Cameroon by Merker et al. in 2021 (7.4%) [25], although it varies depending on geographical context and study populations. Lower rates have been reported in other studies, such as Brian et al. in Malawi (0.3%) [26], whereas higher levels were reported by Ossoga et al. in Chad (12.8%) [7]. Despite the emergence of resistance, isoniazid remains a key drug used both in first-line treatment regimens and in preventive therapy (TPT). This variability may be explained by several factors, including differences in access to rapid diagnostics, treatment adherence, and circulation of resistant strains in certain regions.

For fluoroquinolone resistance (FLQ), mutations were detected in 2 patients, corresponding to a rate of 2.06%. This rate appears relatively low compared to that reported by Mamuda et al. in Nigeria in 2026 (12.5%) [27], but remains lower than values described in 2024 by Nehru et al. in India (5.12%) [28] and Che et al. in China (4.6%) [29]. This difference with highly populated countries may be explained by higher selective pressure due to more widespread and sometimes inappropriate use of fluoroquinolones, both for tuberculosis and other bacterial infections, thereby promoting the emergence of resistant strains. Moreover, differences in surveillance systems and access to drug susceptibility testing may also contribute to these variations. The relatively low rate observed in this study suggests that these drugs remain largely effective in our setting, particularly as second-line treatment options [30].

Among the analyzed samples, rifampicin monoresistance was observed in 1.03% of cases, isoniazid monoresistance in 6.18%, and fluoroquinolone mono- resistance in 1.03%. This result is partially similar to that reported in Chad by Awa et al. in 2017, where rifampicin monoresistance (5.4%) was higher than isoniazid resistance (13.5%) [13]. However, it differs from findings reported in Nigeria by Lana et al. in 2012 (rifampicin 2.8%, isoniazid 1.4%) [24], by Barnett et al. in Malawi (rifampicin 2.9%, isoniazid 0.3%) [26], and by Chan et al. in South Korea in 2012 (rifampicin 9%, isoniazid 4.54%) [31].

Rifampicin monoresistance is relatively rare. However, in this study, 1.03% of patients showed rifampicin monoresistance. The higher proportion of isoniazid resistance (6.18%) among HIV/TB co-infected patients may be explained by immunosuppression and treatment adherence difficulties in these patients. These resistances inevitably require second-line anti-tuberculosis drugs, which are costly in a resource-limited country such as Chad.

Mutations in both rifampicin resistance (rpoB) and isoniazid resistance (inhA or katG) genes of Mycobacterium tuberculosis, defining multidrug resistance (MDR), were detected in 3 samples (3.09%). This rate is close to those reported by Ouédraogo et al. in Burkina Faso (3.8% in 2014) [32], Lana et al. in Nigeria (3.6%) [24], Barnett et al. in Malawi (3.8%) [26], Weyer et al. in South Africa (3.4% in 2007) [33]. However, it is lower than those reported by Doungous et al. in Chad (5,5% in 2026) [34], by Sied et al. in Ethiopia (20% in 2023) [35] and by Chan et al. in Seoul, South Korea (15.9% in 2021) [31], but higher than the 0.9% reported in Chad by Dlinga et al. in 2023 [36]. These variations may be explained by differences in access to rapid molecular diagnosis, drug availability, treatment adherence, and previous treatment history. The presence of resistance among co-infected patients is clinically significant, as it complicates management, increases the risk of treatment failure, and may contribute to transmission of multidrug-resistant strains in vulnerable populations.

Among these, one sample showed additional fluoroquinolone resistance mutations (gyrA and gyrB), corresponding to pre-XDR TB (1.03%). This proportion, although low, remains concerning as it reflects the circulation of highly resistant strains within a vulnerable population, often exposed to difficulties in treatment adherence, prior treatment history, and drug interactions, particularly in the context of HIV–tuberculosis. According to the WHO, pre-XDR forms represent a major public health problem requiring early diagnosis and strengthened surveillance in order to reduce their spread and improve clinical outcomes [5].

A clear male predominance was observed (9M/2F), consistent with findings reported in Ethiopia by Hirpa et al. in 2013 [37], in the DRC by Misombo et al. in 2016 [38], in Gabon by Kombila et al. in 2021 [39], and in Chad by Dlinga et al. in 2023 [36]. Other studies, however, have reported female predominance, such as in Congo by Okemba-Okombi et al. in 2020 [40] and in Ethiopia by Desissa et al. in 2018 [41]. This discrepancy may be partly explained by the composition of our study sample, which included a higher proportion of male participants. Indeed, sex distribution within a study population can strongly influence the observed sex ratio.

In this study, the 35 - 45-year age group was the most affected by multidrug resistance, accounting for more than 60% of cases. This result is similar to that of Kombila et al. in Gabon in 2021, who reported 25 - 45 years in 73.1% of cases [39], and LaFreniere et al. in Canada in 2020, who reported 35 - 44 years in 41.3% of cases [42]. However, it differs from findings in the DRC by Misombo et al. in 2016 (16 - 36 years, 61%) [38] and in Congo by Okemba-Okombi et al. in 2020 (20 - 29 years, 53.84%) [40]. This may be explained by the higher exposure of this age group to risk factors, including repeated antibiotic use and poor treatment adherence, thereby favoring the emergence of multidrug-resistant strains.

In our study, CD4 count below 200 cells/µL observed in patients co-infected with HIV/TB is an important marker of severe immunosuppression and is frequently associated with poor clinical outcomes, particularly in the presence of resistance to anti-tuberculosis drugs. In this study, among the 11 patients presenting resistance to at least one anti-tuberculosis drug, 7 had a CD4 count below 200 cells/µL, corresponding to a rate of 63.6%. This proportion may reflect advanced immunosuppression, which could favor the persistence or emergence of drug-resistant forms of tuberculosis. Several studies have shown that HIV/TB co-infected patients with CD4 counts < 200 cells/µL experience higher mortality and poorer treatment outcomes compared to those with higher CD4 levels. For instance, a study conducted in Benin reported significantly higher mortality among patients with CD4 counts < 200 cells/mm³ compared to those with CD4 counts > 200 cells/µL [43]. Similarly, among patients with multidrug-resistant tuberculosis, low CD4 counts, particularly ≤ 100 cells/µL, were strongly associated with reduced survival and increased risk of death [44]. These findings suggest that severe immunosuppression contributes to worsening tuberculosis progression and may promote treatment failure as well as the development of resistance to anti-tuberculosis drugs.

This study has several limitations. The relatively small sample size may have limited the precision of the estimates. In addition, recruitment was conducted in only two centers in N’Djamena, which may limit the generalizability of the findings to the entire country. Furthermore, only bacteriologically confirmed cases of pulmonary tuberculosis were included, excluding extrapulmonary forms and unconfirmed cases. Finally, drug resistance detection relied solely on the molecular assays Xpert MTB/RIF and Xpert MTB/XDR, without confirmation by phenotypic drug susceptibility testing (DST) or genetic sequencing, which may have affected the estimation of certain resistance patterns.

The non-negligible prevalence of multidrug resistance observed in this study confirms that drug-resistant tuberculosis among HIV/TB co-infected patients remains a major public health challenge in Chad. The most notable finding is the identification of one pre-XDR strain among the 3.09% of MDR cases detected in this study, highlighting the importance of rapid molecular diagnosis to improve therapeutic management and limit the spread of multidrug-resistant Mycobacterium tuberculosis strains.

The authors contributed to the study design, data collection, and manuscript writing, and declare that they have read and approved the final version of the manuscript.

The authors would like to thank the staff of the LNR/PNT, APMS, IRED, and CHU-ATC for their valuable and sincere collaboration.