Bridging the gap in TB diagnostic strategies between technical sophistication and real-world applicability in low-resource settings

Dear Editor,

We thank Barr et al.¹ for their recent thoughtful engagement with our published work² and appreciate their comments. We have previously reviewed their work on needle emulsification of Mycobacterium tuberculosis (MTB) and the use of flow cytometry-based counting of total bacilli, an elegant approach that has contributed meaningfully to the field.³ However, our methodological approach – including the use of a BSL-2 strain, our spiking experiment protocol, and a more scalable pre-processing method – was deliberately chosen to prioritize reproducibility in resource-limited settings. Furthermore, the use of colony-forming-unit (CFU) counting, despite its acknowledged limitations, aligns with historical methods that have been foundational in TB diagnostics and remains a broadly applicable approach. We agree that flow cytometry offers enhanced precision for bacillary quantification; however, its application is limited in resource-limited settings where the burden of disseminated TB is highest. Our goal was to prioritize a method that could be more feasibly deployed at scale in these settings.

While we certainly agree that the proportion of non-culturable MTB bacilli may vary depending on culture conditions, this does not diminish the clinical significance of a positive molecular test in a critically ill patient. CFU enumeration will inherently underestimate the cumulative nucleic acid signal from MTB, as DNA may originate from multiple sources, including viable CFUs, non-culturable but metabolically active MTB, metabolically inactive MTB, and cell-free MTB DNA. This limitation is not unique to spiked culture experiments; rather, it remains an inherent challenge when testing actual clinical specimens as well. For example, clinical samples from patients with severe disseminated TB treated with early anti-TB therapy may still yield strong MTB DNA signals by molecular testing, even if culturable bacilli are absent.

From a clinical perspective, the primary issue is sensitive detection of disseminated MTB in critically ill persons living with HIV (PLWH), as prompt diagnosis is paramount to therapeutic decision-making. Precise bacterial quantification, particularly at the lower limits of detection, is secondary to the overarching goal of reliably identifying disseminated TB in a manner that is both operationally feasible and clinically actionable. Xpert MTB/RIF Ultra and related molecular diagnostic assays provide a significant advance in this regard, enabling the detection of MTB bloodstream infection where more sophisticated techniques such as flow cytometry are not available.

We emphasize that our approach sought to bridge the gap between technical sophistication and real-world applicability, prioritizing diagnostic strategies that may be implementable in high-burden, low-resource settings. Given these constraints, rather than focusing on absolute quantification, our emphasis remains on maximizing translational impact and facilitating timely initiation of appropriate therapy in vulnerable, critically ill persons.