In this study, we detected an unexpectedly high prevalence of M. tuberculosis DNA in 12 to 16% of respiratory samples from predominantly US-born patients hospitalized in the principal safety net hospital in Boston with a variety of infectious and non-infectious syndromes. While unforeseen and preliminary, these results are plausible given: (1) TOP assay’s established diagnostic accuracy against a composite reference standard in over 400 patients with presumptive pulmonary TB from Uganda, Brazil and the US^19,20; (2) the strength of our laboratory methods that demonstrate robust analytical sensitivity, and analytical specificity supported by DNA amplicon sequencing in both in-vitro conditions and clinical studies; (3) relatively high TOP optic density values (median 0.520) using a conservative cut-off, making false positive readings unlikely; (4) evidence of reproducibility by closely replicating both the point prevalence estimate, and the genetic distribution of M. tuberculosis 2ponA variants in two independent study samples obtained almost 1 year apart; (5) evidence of specificity by detecting a markedly lower positivity rate (2%) in a comparable cohort with a presumably lesser TB burden; (6) finding a clear bimodal distribution of positive results according to age—a key epidemiologic parameter of TB disease in the US⁴, and; (7) presence of known social, clinical and laboratory indicators of TB disease in TB-DNA+ individuals. Pending confirmation from larger multicenter studies, these findings open the possibility of an association—etiological, mechanistic, or non-causal- between M. tuberculosis and a variety of clinical syndromes in patients with a low pre-test probability of TB in the US.

The natural history of TB disease is a continuum that begins with the establishment of M. tuberculosis infection following an infectious exposure^6,11. In individuals that are unable to contain bacterial replication, early disease progression occurs through consecutive asymptomatic or pauci-symptomatic inflammatory stages that are mostly culture-negative, eventually culminating in symptomatic illness that is usually confirmed by mycobacterial cultures^{6,7,9,11,12,13,14,16,21,22}. However, bacterial replication kinetics are variable, often following one of several possible paucibacillary trajectories over time, including persistently low bacterial load, delayed progression, and even self-cure^9,12,14,16. Recently developed molecular assays approved by the WHO (e.g., Xpert MTB/RIF Ultra and TrueNat) have a sensitivity comparable to culture and have significantly shorten the “sample-to-treatment” cascade of care, but they are still best suited for the detection of TB disease at later stages and have had limited impact in improving treatment outcomes²³. Responding to a need for more sensitive tools to identify early stages of TB disease^2,5,21, recent development efforts have focused on blood-based gene signatures and inflammatory biomarkers that have been tightly calibrated against culture-confirmed TB disease. However, these biosignatures have demonstrated sub-optimal diagnostic accuracy and reproducibility problems across populations^2,10,21,22. In contrast, molecular detection methods with sensitivity superior to culture, such as the TOP assay, have a long track record of delivering clinically useful results, often replacing culture as the preferred diagnostic modality for multiple organisms and sample types²⁴. This study provides further impetus to reconsidering the standard practice of anchoring new TB diagnostic studies to a microbiological yardstick defined by culture, as such evaluations may be flawed by design^3,20,21. This focus on demonstrating bacterial viability with cultures (or other means) as a prerequisite to defining clinically relevant TB disease also limits the interpretation of our results within the framework of the recent “International Delphi Consensus Classification of Early Tuberculosis States”^17,18.

While certainly unforeseen, several results from the present study align well with currently available knowledge. First, 75% of TB-DNA+ patients were 50 years or older, a finding that is consistent with the epidemiology of TB in the US, where the rate of TB disease is highest in older individuals, and 85% of new TB cases are related to reactivation from a remote exposure^4,25,26. This result may be best understood by applying the “iceberg principle,” where this ultrasensitive assay is simply detecting additional TB cases that are currently hidden below the level of detection of current technologies. Similarly, the clinical description of the TB-DNA+ adolescent is consistent with unstable TB infection or early TB disease^6,11, as he first presented to BMC when he was 27.

Consequently, upon careful consideration, the most unexpected finding of this study is the detection of M. tuberculosis DNA in the sputum of the only three patients with sickle cell disease that shared a diagnosis of acute chest syndrome (3/16 vs. 0/85, p = 0.003), a finding that should be considered preliminary given the small sample size despite strong statistical significance. The increased risk of infectious complications in patients with sickle cell is well established, particularly when complicated by acute chest syndrome^28,29. In their seminal study, Vichinsky et al. found that among 671 episodes of acute chest syndrome, 216 (32%) were caused by infections from 27 different microorganisms, predominantly atypical bacteria and viruses, but 2 (0.3%) cases were attributed to M. tuberculosis²⁸. Importantly, in 46% of patients, the etiology of acute chest syndrome was undetermined (e.g., no evidence of infection, fat embolism, or infarction); in the present study, all three patients had a negative concomitant microbiological work-up. Although US populations at risk for TB and those at risk for sickle cell disease may have overlapping demographic and social characteristics, a definitive link between these two illnesses has not been established²⁸. If confirmed in larger studies, this previously unsuspected clinical association may be particularly relevant to settings where both maladies are more prevalent, such as in sub-Saharan Africa²⁹.

After a dramatic decline in the number of TB cases in the US over the last 25 years, there is growing evidence the epidemic has entered a period of non-declining disease rates⁴, particularly following the COVID period⁴. Complicating elimination, 87% of new cases arise from the large reservoir of individuals with untreated TB infection⁴. When individuals with TB infection escape latency, establishing a diagnosis of TB disease is often challenging because of evolving risk factors, atypical clinical presentations following immunotherapy, and a growing need for detection of culture-negative disease^2,9,13,16. In 2022, 21% of TB cases reported to CDC were not microbiologically confirmed (e.g., clinical TB case definition with no M. tuberculosis viability demonstrated), and this proportion has slowly increased over time due to epidemiological and biological factors^4,13,19. Interestingly, most TB-DNA+ patients in this study were TST or IGRA-negative, a poorly understood condition that has been associated with advanced age, low peripheral lymphocyte counts, and poor clinical outcomes in microbiologically confirmed TB cases in the U.S. and elsewhere^30,31. This observation is also frequently reported in presumptive TB patients that are Xpert MTB/RIF Ultra trace-positive, a semi-quantitative diagnostic category measuring very low bacillary loads that are often culture-negative³².

Currently, isolated detection of M. tuberculosis DNA is not universally accepted as evidence of TB disease²⁰ but this opinion is evolving with the growing body of evidence provided by presumptive TB patients that are Ultra trace-positive³³. Although preliminary, the results of this study lead to several mechanistic, clinical, and infection control questions that will require further investigation to elucidate. First, detection of M. tuberculosis DNA in certain patients may be clinically inconsequential, as it may be capturing non-viable bacteria or represent shedding from a latent focus⁸. Yet, disregarding evidence of M. tuberculosis DNA in respiratory samples without further study is surely unwise. Second, if linear disease progression culminating in culture-positive illness is indeed the common final stage of TB, our findings may suffer from insufficient follow-up time to culture-positive conversion. An alternative explanation is that disease progression was interrupted or modified by partial treatment with beta-lactam antibiotics (81% of TB-DNA+ patients received them), given their well-described antituberculous activity³⁴. Finally, we hypothesize that this new diagnostic tool may have permitted the discovery of a previously undetectable variant of paucibacillary TB disease that stands as a polar opposite to the traditional, culture-positive multibacillary form of the disease. This presumed bipolar TB pathogenesis model that has been proposed by others³⁵, would have a strong biological precedent, as shown by other closely related mycobacteria such as M. leprae³⁵ and certain nontuberculous mycobacteria³⁶. One possible mechanistic explanation for this bipolar spectrum of TB disease is our household contact study from Brazil, showing that less transmissible M. tuberculosis clinical isolates cause diffuse inflammation in C3HeB/FeJ mice lung pathology when compared to highly transmissible strains that exhibited more caseating granulomas, a lesion type with high potential to cavitate and therefore cause multibacillary TB disease³⁷.

Our study has limitations. The TOP TB assay is a research-use-only tool that currently lacks regulatory clearance. Also, our results are challenged by the absence of a confirmatory independent test, as currently available diagnostic methods do not have the requisite sensitivity to detect the very low bacterial loads ascertained by the TOP assay (i.e., culture, other molecular assays and metagenomic sequencing), or because they depend on M. tuberculosis growth in culture to sufficiently enrich the molecular target (i.e., whole-genome sequencing). The use of discarded respiratory samples in all three studies is not ideal because it may have affected the quality and volume of specimens, and risk sample contamination. Whereas the likelihood that cross-contamination or insufficient assay specificity may have resulted in false-positive results is low, it cannot be completely excluded. Because of the current diagnostic uncertainty of detecting M. tuberculosis DNA in culture-negative samples, we cannot certify M. tuberculosis directly contributed to symptoms, pathology, or death in the absence of tissue or autopsy results. The latter limitation also needs to be considered when interpreting the positive predictive value of the TOP assay, given its heightened sensitivity and low pre-test probability of disease in the study cohort. Finally, while passive follow-up through serial chart review provided detailed and reliable information, some additional characteristics might not have been identified. Future prospective clinical studies with dedicated respiratory specimens will be necessary to confirm and expand our findings.

In summary, we detected a higher-than-expected prevalence of M. tuberculosis DNA in respiratory samples of predominantly US-born patients hospitalized in a Boston safety-net hospital. Whereas most TB-DNA+ patients were older and presented with a variety of infectious and non-infectious syndromes commonly encountered in hospitalized individuals, there was also a group of four young patients, three of which shared the diagnosis of acute chest syndrome—a striking and potentially consequential clinical association. While preliminary, we hypothesize that our findings indicate the existence of a paucibacillary form of TB that remains unrecognized and is not detectable using current diagnostic tools. These results will require confirmation in larger prospective studies that include clinical, radiological, immunological, and microbiological correlation. Yet, even without complete knowledge of biological mechanisms, impact on clinical outcomes, or transmissibility, the evidence presented supports dissemination given the potential implications for medical care and public health in the US, and elsewhere.