Tuberculosis (TB) remains one of the major infectious diseases worldwide, representing a significant public health problem, particularly in sub-Saharan Africa 12. According to the latest World Health Organization, 2024 report, approximately 10.8 million people developed TB in 2023, with 1.25 million deaths, including a substantial proportion among people living with HIV 1. Early and accurate detection of Mycobacterium tuberculosis (MTB) is crucial to reduce TB-related morbidity and mortality. Diagnostic methods in use include smear microscopy, molecular tests such as Xpert MTB/RIF, and culture, which is considered the gold standard due to its high sensitivity and ability to provide a definitive diagnosis 34.

For decades, direct smear microscopy has been the most widely available initial diagnostic test in resource-limited settings such as the Republic of Congo 5. In this country, TB is also a major concern, with cases of multidrug-resistant TB (MDR-TB) and frequent HIV co-infections, complicating disease management and control 67. While its technical simplicity and low cost are operational advantages, its notoriously low sensitivity, particularly in HIV patients or those with paucibacillary disease, likely results in a substantial number of missed diagnoses 8. However, the technical complexity, long turnaround time, and biosafety requirements of culture limit its utility for rapid clinical decision-making at the point of care 3.

To bridge this diagnostic gap, the WHO recommends the Xpert MTB/RIF assay as the initial diagnostic test for TB because of its ability to rapidly detect MTB complex DNA and rifampicin resistance 49. In the Republic of Congo, this recommendation has driven a significant shift in diagnostic policy, with Xpert now widely adopted as the first-line diagnostic tool in more than 40% of testing centers 7.

However, critical reliance on microscopy persists for patient follow-up to monitor treatment response, even in centers equipped with GeneXpert. Moreover, in many centers, microscopy remains the only on-site tool for both initial diagnosis and follow-up. This continued, widespread reliance on direct smear microscopy for both initial diagnosis and treatment monitoring underscores the need for a contemporary evaluation of its performance in the Congolese context, especially in high-risk populations such as people living with HIV 5610. Generating such local evidence is essential to validate current practices, identify potential risks, and guide the evolution of national TB diagnostic strategies.

This study evaluated the diagnostic performance of Xpert MTB/RIF assay and smear microscopy in a cohort of Congolese patients with presumptive pulmonary tuberculosis hospitalized at the Makelekele Hospital, using culture as the gold standard. Our patient demographic, predominantly younger adults, reflects the typical population most affected by TB 11415. Clinically, the same observation was made regarding common symptoms such as chronic cough, HIV infection, and fever 16177. The overall findings confirm that while smear microscopy remains highly specific, its sensitivity is considerably lower than that of Xpert MTB/RIF, underscoring the limitation of microscopy as a primary diagnostic tool 182.

Smear microscopy detected fewer MTB cases than culture (32% vs 49%), with a sensitivity of only 57.8%. This means that over 40% of true TB cases confirmed by culture were missed by microscopy. The low sensitivity directly translates into a modest Negative Predictive Value (NPV) of 69.4%, which is a critical weakness in high-burden settings such as the Republic of Congo. This modest NPV indicates that if diagnosis relies solely on microscopy, a substantial proportion of true TB cases may be missed. The finding underlines the limited reliability of smear microscopy in high-burden settings, particularly where early and accurate detection is essential for reducing transmission 192.

In contrast, the Xpert MTB/RIF assay demonstrates a markedly improved diagnostic profile. Xpert MTB/RIF detected more MTB cases than culture (62% vs 49%) and achieved excellent sensitivity (97.8%) while maintaining a high specificity (77.2%). The high negative predictive value (97.1%) suggests that Xpert MTB/RIF is highly reliable in excluding TB in negative cases, a critical advantage in clinical practice. The apparent higher detection rate compared with culture may be due to the molecular assay’s ability to detect DNA from non-viable bacilli or culture losses due to sample degradation or contamination. Despite this, the strong statistical association between Xpert MTB/RIF and culture results supports its validity as a diagnostic tool 20.

The relatively low sensitivity of microscopy observed in our study compared to Xpert MTB/RIF is consistent with several previous studies. Steingart et al. 821 showed that the sensitivity of Ziehl–Neelsen smear microscopy for the detection of MTB ranges between 50% and 60%, whereas the sensitivity of Xpert MTB/RIF can exceed 90%, particularly among HIV co-infected patients. This highlights the limitation of microscopy in detecting paucibacillary cases, which may go unnoticed, while Xpert MTB/RIF, based on PCR amplification, efficiently detects MTB genetic material even at low bacillary loads 1222239.

Taken together, these findings reinforce the WHO recommendation that Xpert MTB/RIF should be prioritized as the initial diagnostic test for TB 624, especially in high TB burden and resource-limited settings. While microscopy remains useful because of its simplicity, rapid turnaround time, and low cost, its low sensitivity significantly limits its standalone diagnostic value. Culture remains indispensable for definitive confirmation but is constrained by longer turnaround times and higher infrastructure requirements 311.

This study reinforces the pivotal role of molecular diagnostics in the modern TB care cascade. While smear microscopy retains utility due to its high positive predictive value and ability to indicate infectiousness, its critically low sensitivity remains a major limitation. In contrast, the Xpert MTB/RIF assay has proven to be a far more reliable tool, offering significantly superior sensitivity and an exceptional ability to rule out disease. Its implementation is indispensable for strengthening TB control efforts, as it facilitates earlier and more accurate diagnosis, enables prompt treatment initiation, and ultimately curbs community transmission.

Therefore, these findings provide robust, local evidence to support the multiple appeals made to national health authorities to equip all TB screening centers across the Republic of Congo with GeneXpert technology.

FN and DOEA conceived the study. DOEA and MAEN contributed to study design and sample processing or methodology. FHM, JCV, FHOO analysed the data. FN had overall responsibility for the study. AG, FHOO, FHM, and FN were involved in conceptualization, data compilation, editing, and review of drafts, as well as finalization. All authors contributed to drafting the manuscript and read and approved the final version

This study received financial support from CANTAM (EDCTP-RegNet2015-1045).