This prospective longitudinal study was conducted from March 2023 to September 2025 at Jamot Hospital in Yaoundé, Cameroon’s national reference centre for tuberculosis management. The study adhered to STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines.

Ethical approval was obtained from the Regional Ethics Committee for Human Health Research, Central Region (CERSHC), under approval number CE N˚1297/CRERSHC/2022, dated 19 September 2022. The study was conducted in strict accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Written informed consent was obtained from all participants. Confidentiality was maintained through numerical coding of all data.

The target sample size was calculated a priori based on the nationally representative estimate of rifampicin resistance (RR-TB, proxy for MDR-TB) among new bacteriologically confirmed pulmonary TB cases in Cameroon reported by Noeske et al. (2018): a RR-TB prevalence of 1.6% (95% CI: 0.8% - 2.3%) [13]. This figure was adopted as the reference prevalence for the present sentinel-site study. Using the standard single-proportion formula n = z² × p (1 − p)/e², with z = 1.96 (two-sided 95% CI), p = 0.016, and a desired absolute precision of e = ±2.6 percentage points, the minimum evaluable sample size was: n = (1.96)² × 0.016 × 0.984/(0.026)² ≈ 90 patients.

Consecutive non-probability sampling of eligible treatment-naïve TB patients presenting at Jamot Hospital was used. All eligible patients were approached sequentially from March 2023 until the target was reached. The extended recruitment period captured seasonal and temporal variations in TB epidemiology.

Inclusion criteria: Age ≥ 21 years; sputum smear-positive by auramine or Ziehl-Neelsen microscopy; no documented prior anti-TB treatment (verified through patient interview, medical records review, and national TB programme registry check); residence within 50 km of Yaoundé to facilitate follow-up.

These criteria were selected to characterise primary resistance in the highest-burden diagnostic subgroup accessible at this referral centre. The lower age limit of 21 years reflects minimum legal consent age under Cameroonian regulations and the dominant TB demographic in urban Yaoundé. Smear-positivity was required to ensure sufficient bacillary load for reliable MGIT culture and MTBDRplus genotyping. Residence within 50 km was necessary to achieve 6-month follow-up completeness. Accordingly, the findings are generalisable to smear-positive, treatment-naïve adults ≥ 21 years residing near Yaoundé and should not be extrapolated to paediatric cases, smear-negative disease, or patients residing in rural or remote areas.

Exclusion criteria: Extrapulmonary TB only; HIV, hepatitis B, or hepatitis C co-infection (verified by rapid diagnostic testing); ongoing immunosuppressive therapy; baseline serum creatinine > 120 μmol/L or ALT/AST > 2 × upper limit of normal; inability or unwillingness to provide informed consent or complete 6-month follow-up.

The exclusion of HIV, HBV, and HCV co-infected patients was motivated by the differential influence of co-infection on resistance rates and treatment outcomes, which would have introduced important confounding. These immunocompromised patients represent a distinct subgroup warranting dedicated investigation. Their exclusion limits the generalizability of resistance estimates given Cameroon’s high HIV-TB co-infection rate (29%) and should be acknowledged as a key limitation.

Figure 1 presents the complete participant flow. Briefly, 147 patients were screened for eligibility during the study period. Of these, 42 were excluded: 12 reported prior anti-TB treatment, 9 were HIV-positive, 7 had HBV or HCV co-infection, 6 resided > 50 km from Yaoundé, 4 had extra pulmonary TB only, 2 had abnormal renal/hepatic function, and 2 refused consent. The remaining 105 patients were enrolled. Of these, 100 (95.2%) yielded positive MGIT cultures; 5 remained culture-negative and were excluded from resistance analyses. Among the 100 culture-positive isolates, 98 (98.0%) were successfully genotyped by MTBDRplus (2 yielded indeterminate results due to insufficient DNA). All 105 enrolled patients completed the 6-month treatment follow-up.

Sociodemographic, clinical, and microbiological data were collected prospectively using a standardised case report form. An early-morning sputum specimen (~2 mL) was collected from each enrolled patient and transported within 4 hours at 4˚C to the TB Research Laboratory, Biotechnology Centre of Nkolbisson, University of Yaoundé I.

Figure 1. Participant flow. Of 147 patients screened, 42 were excluded (prior treatment, HIV infection, HBV/HCV co-infection, distant residence, extrapulmonary TB, renal/hepatic impairment, or refusal). A total of 105 patients were enrolled; 100 were culture-positive and 98 successfully genotyped. All enrolled patients completed 6-month follow-up.

Auramine O Fluorescence Microscopy: Sputum smears were stained using the standard auramine O fluorochrome method and examined at 400× magnification. Smear positivity was graded according to WHO standards: scanty (1 - 19 AFB per slide length), 1+ (20 - 199 AFB), 2+ (5 - 50 AFB per field), or 3+ (>50 AFB per field) [14][15].

MGIT Culture: Mycobacterial culture was performed using the BACTEC™ MGIT™ 960 automated liquid culture system (Becton Dickinson). Sputum samples were decontaminated using the NaCl-NaOH method, neutralized with 67 mM phosphate buffer, and centrifuged at 3000 – 4000 × g for 15 minutes at 4˚C. The pellet (0.5 mL) was inoculated into MGIT tubes with Middlebrook 7H9 broth, OADC enrichment, and PANTA antibiotic mixture. Automated fluorescence monitoring was performed from day 14 through day 42. Positive cultures were confirmed as M. tuberculosis complex by colony morphology, acid-fast smear, and MTBDRplus [16].

GenoType®MTBDRplusAssay: DNA was extracted from MGIT-positive cultures by standardised thermal lysis. Multiplex PCR targeted rpoB, katG, and the inhA promoter [13][17]. Amplicons were hybridized to nitrocellulose strips containing 27 oligonucleotide probes. Strip interpretation followed the manufacturer’s reading guide and was cross-validated by two independent observers.

Baseline DST results were communicated to the treating clinician within 48 hours of genotyping. Patients with fully susceptible isolates were maintained on the standard WHO 2HRZE/4HR regimen. Patients with rifampicin mono-resistance were referred for individualised regimen adjustment in accordance with national MDR-TB guidelines; patients meeting MDR-TB criteria received WHO-recommended MDR-TB regimens. Because resistant cases were moved to alternative regimens, sputum conversion data are reported separately by baseline resistance status to avoid confounding treatment-outcome comparisons.

Data were analysed using IBM SPSS 23.0 and R 4.2.0. Categorical variables are reported as frequencies and percentages with 95% Wilson score CIs; continuous variables as medians with IQRs. Drug resistance prevalence was estimated with exact binomial 95% CIs. Between-group comparisons used Fisher’s exact test and Mann-Whitney U test. Sputum conversion rates were compared by Kaplan-Meier analysis with log-rank tests. All tests were two-tailed (p < 0.05).

A total of 105 treatment-naïve pulmonary tuberculosis patients were enrolled at Jamot Hospital between March 2023 and September 2025 and were completed the 6-month treatment follow-up.

The cohort was predominantly male (73.3%, n = 77) with a median age of 34 years (range: 21 - 65 years). Baseline bacillary burden was substantial, with 70.5% (74/105) exhibiting high bacterial loads: 42.9% (n = 45) graded 2+ and 25.7% (n = 27) graded 3+. Lower-grade positivity accounted for the remainder: 17.1% (n = 18) scanty and 14.3% (n = 15) graded 1+ (Table 1).

Table 1. Distribution of baseline sputum smear grades.

Viable mycobacterial growth was detected in 95.2% (100/105) of baseline specimens. The five culture-negative cases (4.8%) likely represented non-viable bacilli, pre-analytical errors, or contamination during processing, as all were smear-positive.

Sputum conversion data are presented separately by baseline drug susceptibility status, since patients with drug-resistant isolates were switched to alternative regimens (see Section 2.7). Among the 66 fully susceptible patients who continued standard 2HRZE/4HR therapy, rapid bacteriological clearance was observed: 88.6% converted to smear-negative by month 1, increasing to 94.3% by month 2, and 96.2% (63/66) achieved sustained sputum conversion at treatment completion (month 6). Three patients (4.5% among susceptible cases) remained persistently smear-positive at month 6, raising concern for undetected resistance to pyrazinamide or ethambutol, poor adherence, or pharmacokinetic variability. For the 32 patients with resistant isolates, treatment outcomes following regimen adjustment are reported descriptively: all 14 rifampicin mono-resistant and all 7 MDR-TB patients were referred to specialised MDR-TB management; follow-up data from those programmes were not available within the timeframe of this study and are therefore not included in the overall cure-rate estimate (Figure 2).

Note: Analysis limited to patients on standard 2HRZE/4HR; drug-resistant cases (n = 32) were excluded.

Figure 2.Sputum smear conversion during standard therapy. In fully drug-susceptible pulmonary TB patients (n = 66), smear conversion reached 88.6% (M1), 94.3% (M2), and 96.2% (M6); 4.5% remained positive at treatment completion.

Among the 100 culture-positive isolates, 98 were successfully genotyped using the GenoType MTBDRplus assay. Drug resistance was detected in 32.7% (32/98) of isolates: 14 isolates (14.3%) exhibited rifampicin mono-resistance, 11 (11.2%) showed isoniazid mono-resistance, and 7 (7.1%) met criteria for MDR-TB. The remaining 67.3% (66/98) were fully susceptible (Table 2).

Table 2. Drug resistance profiles among genotyped M. tuberculosis isolates.

RIF = rifampicin; INH = isoniazid; -S = susceptible; -R = resistant.

Twenty-one rifampicin-resistant isolates were analyzed for rpoB gene mutations within the RRDR (codons 507 - 533). Three mutations accounted for all detected resistance: rpoB H526Y and H526D (probe MUT2B) were equally prevalent at 38.1% (8/21) each, while rpoB S531L (MUT3) was identified in 23.8% (5/21) of cases (Table 3). All three mutations confer high-level rifampicin resistance by disrupting rifampicin binding to the β-subunit of RNA polymerase.

Eighteen isoniazid-resistant isolates harboured mutations in katG or the inhA promoter. The katG S315T mutation predominated: S315T2 was detected in 55.6% (10/18) and S315T1 in 22.2% (4/18), collectively accounting for 77.8% of isoniazid resistance. The inhA promoter mutation C-15T was identified in 22.2% (4/18) of isolates (Table 4).

Table 3. Distribution of rpoB mutations conferring rifampicin resistance.

Table 4. Distribution of katG and inhA mutations conferring isoniazid resistance.