The long-term risk of tuberculosis among individuals with Xpert Ultra “trace” screening results: a longitudinal follow-up study

  1. Joowhan Sung
  2. Mariam Nantale
  3. Annet Nalutaaya
  4. Patrick Biché
  5. James Mukiibi
  6. Joab Akampurira
  7. Rogers Kiyonga
  8. Francis Kayondo
  9. Michael Mukiibi
  10. Caitlin Visek
  11. Caleb E Kamoga
  12. David W Dowdy
  13. Achilles Katamba
  14. Emily A Kendall

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Abstract

Background

Systematic screening for tuberculosis using Xpert Ultra generates “trace” results of uncertain significance. Additional microbiological testing in this context is often negative, but individuals with trace results might have early disease or elevated risk of tuberculosis.

Methods

We screened for tuberculosis with Xpert Ultra in Uganda, enrolling individuals with trace-positive results and Ultra-negative controls. Participants without tuberculosis on extensive initial evaluation were followed, with repeat testing at 1, 3, and 6 months after trace results, and at 12 and 24 months for all participants. We estimated cumulative cause-specific hazards of incident tuberculosis, considering a definition of tuberculosis that included clinician judgment and one based strictly on microbiological results. We compared participants with Ultra-trace versus Ultra-negative sputum, and subgroups of participants with Ultra-trace sputum.

Findings

Of 129 participants with trace-positive screening results, 45 (35%) were recommended for treatment upon enrollment, and eight were lost to follow-up within three months. Of 76 remaining participants followed for median 697 (interquartile range 179-714) days, 20 (26%) were recommended for tuberculosis treatment. The cumulative hazard of clinician-defined incident tuberculosis was 26% (95% confidence interval: 14-38%) at one year and 35% (19-52%) at two years, versus 2% (0-5%) at two years for controls. Hazards were similar for microbiologically defined incident tuberculosis. Incident tuberculosis was strongly associated with abnormal baseline chest X-ray (hazard ratio 15.0 [3.4-65.1]) but not with baseline symptoms.

Interpretation

Individuals with trace-positive sputum during screening, particularly those with abnormal chest imaging, are at substantial risk of incident tuberculosis over the subsequent two years.

Funding

National Institutes of Health

Research in context

Evidence before this study

Recent advances in tuberculosis research have shifted the disease framework from a binary classification of latent versus active tuberculosis to a continuum of disease states. They have also led to a better understanding of the dynamic disease course of early tuberculosis, which can either progress to culture-positive disease or regress spontaneously over time. “Trace” results from Xpert MTB/RIF Ultra (“Ultra”) are sometimes perceived as false positives in individuals who subsequently test negative on additional diagnostic assays. However, some of these individuals may have early tuberculosis that falls below the detection threshold of existing diagnostic tests and could progress to microbiologically detectable disease over time. To investigate this, we searched PubMed for studies published up to February 7, 2025, using the terms “tuberculosis” AND (“Xpert OR “Xpert Ultra” OR “Ultra”) AND “Trace” and also reviewed the reference lists of relevant search results. Two prevalence surveys that used Xpert Ultra as a confirmatory test for individuals with symptoms or an abnormal chest X-ray found that 20% and 46% of those with trace-positive sputum had positive cultures. In a study conducted in Uganda where Ultra was used as an initial screening test, only 14% of individuals with a trace-positive result had positive sputum cultures. However, no prior studies have prospectively examined the incidence of tuberculosis among individuals with a trace-positive Ultra result during systematic screening who are otherwise microbiologically negative and not started on treatment.

Added value of this study

In this study, individuals with Ultra trace-positive screening results who were not started on treatment after extensive diagnostic testing were followed for up to two years with repeated testing. About 25% developed tuberculosis during follow-up, and the 2-year cumulative hazard of incident tuberculosis was substantial at 35% (95% confidence interval 19-52%). Those who had a normal chest X-ray at enrollment were at significantly lower risk of developing tuberculosis. Incident tuberculosis risk was similar between those who reported symptoms at the time of enrollment and those who did not.

Implications of all the available evidence

The high incidence of tuberculosis observed among people with trace results in this study support provision of treatment to most individuals who receive trace results during tuberculosis screening. These results also demonstrate that X-ray could be a useful tool to guide treatment decision-making for individuals with trace-positive sputum.

Article activity feed

  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/15486302.

    The long-term risk of tuberculosis among individuals with Xpert Ultra "trace" screening results: a longitudinal follow-up study 

    1. Summary and Overall Impression

    Recommendation: Minor revisions are required. 

    Tuberculosis (TB) remains the leading single-agent infectious cause of death globally, and early detection could reduce transmission and improve outcomes, especially in high-burden settings. The longitudinal cohort study by Sung et al. investigates the long-term risk of TB among individuals identified with Xpert MTB/RIF Ultra "trace" sputum results through community-wide screening in Kampala, Uganda. "Trace" results indicate extremely low levels of Mtb DNA and often go microbiologically unconfirmed, but can be biologically relevant and indicative of a need to consider empiric treatment. Participants with trace-positive results underwent extensive baseline evaluation, including molecular testing, culture, HIV testing, chest X-rays (CXR), and chest CTs. Individuals without a microbiological confirmation or clinical diagnosis of TB at baseline were prospectively followed for up to two years with serial microbiological and radiographic assessments. Among 129 individuals with trace-positive screening results, 35% were diagnosed with TB at baseline. Of the 76 individuals not referred to treatment at baseline, 26% subsequently developed TB over two years, yielding a cumulative hazard of 35% (95% CI: 19-52%). This was markedly higher than the 2% observed in Ultra-negative controls. Incident TB risk was strongly associated with abnormal CXR at baseline (HR 15.0, 95% CI: 3.4-65.1) but showed no correlation with symptom status, HIV infection, sex, or prior TB history. Computer-aided detection (CAD) of baseline CXR showed predictive performance (AUC 0.78-0.85) for identifying individuals who later progressed to TB, particularly in those without prior history of TB. The findings challenge the perception that Ultra trace results represent false positives in active case finding or community screening contexts, but methodological weaknesses must be addressed prior to publication. Sung et al. present compelling evidence of an increased risk of progression to active TB in trace-positive people, particularly when combining trace results with CXR. The authors advocate for consideration of empiric TB treatment initiation or preventative therapy for individuals identified as "trace"-positive during screening, but the recommendation to treat most trace-positive individuals requires further validation through future studies.

    1. Section-by-Section Review

    Methods

    Subheading: Symptom-Neutral Tuberculosis Screening and Participant Recruitment

    Major issues: 

    1. The manuscript mentions that negative controls were age- and sex-matched, but it lacks crucial details on the matching procedure. We recommend that the authors provide complete details of the matching methodology, including whether individual or frequency matching was used, the matching ratio, and the algorithm for selecting matches. Describe any post-matching statistical adjustments performed to account for residual imbalances in known TB risk factors. Address how matching on only age and sex might impact the validity of between-group comparisons. Consider performing sensitivity analyses using propensity score methods to account for potential selection bias. 

    Subheading: Evaluation and Follow-Up of Study Participants

    Major issues:

    1. The manuscript should clarify blinding procedures for the physician panel making treatment recommendations, which constitute the primary outcome measure. While radiologists were explicitly described as "blinded to clinical information," there is no mention of whether the physician panel members were blinded to the initial Ultra trace result when determining if a participant should receive TB treatment. If consultants were not blinded, acknowledge this as a limitation and discuss how it might have influenced treatment decisions.

    2. The implementation of substantially different follow-up protocols between trace-positive participants and controls warrants careful consideration, as it introduces a risk of surveillance bias. Trace-positive participants underwent comprehensive evaluations at 1, 3, 6, 12, and 24 months, while negative controls received only "symptom assessment at 6 months and repeat sputum testing at 12 and 24 months." This differential intensity of surveillance might artificially inflate the relative incidence in the trace-positive group by detecting cases that would have remained undiagnosed with less intensive monitoring. The ascertainment of the outcome (tuberculosis diagnosis) depends partly on the intensity of follow-up, which differed systematically between exposure groups. This methodological issue is particularly concerning for the primary analysis comparing the cumulative incidence between groups. Please acknowledge the differential follow-up as a study limitation and discuss its potential impact on the hazard ratio estimates. Consider performing a sensitivity analysis restricting outcome ascertainment in trace-positive participants to the same timepoints used for controls (6, 12, and 24 months). Discuss how the effect size might be adjusted to account for this surveillance bias.

    Minor issues:

    1. The study would benefit from enhanced clarity regarding the standardized criteria used by the physician panel for making treatment recommendations. Explicitly describe the criteria or decision algorithm that guided treatment recommendations, including how consultants weighed different types of evidence (clinical, radiological, microbiological). 

    Subheading: Statistical analysis

    Major issues:

    1. The statistical approach appears to lack multivariable adjustment to control for the confounding factors that were measured. While the authors report hazard ratios for individual risk factors (e.g., abnormal CXR, HR 15.0), this univariate approach fails to isolate the independent effect of trace-positive status from other measured risk factors. Conduct and report multivariable Cox regression analyses, adjusting simultaneously for all measured potential confounders. Perform formal interaction testing for effect modification by baseline CXR status and other key variables. Consider presenting stratified hazard ratios for trace-positive status across different subgroups (e.g., normal vs. abnormal CXR). 

    Results

    Major issues:

    1. Line 194 and Figure 2 report that n=129 participants with trace-positive sputum (PWTS), which is misaligned with n=121 in Table 1, as well as the header of Table 2, which indicates "Trace Ultra (n=123)". The subgroups of the "Trace Ultra (n=123)" sum to 117, not 123. If these discrepancies are due to attrition, please indicate this (e.g., why n=123 in Table 2 differs from n=129 elsewhere). 

    2. The study identifies a strong effect modification by baseline CXR status (HR 15.0, 95% CI 3.4-65.1) but would benefit from incorporating this finding into a more comprehensive analytic framework. This substantial interaction suggests that the value of trace-positive results varies dramatically depending on CXR findings, which has critical implications for clinical decision-making. Without any formal interaction testing, it remains unclear whether the broad recommendation to treat most individuals with trace-positive results is appropriate for all subgroups, particularly those with normal chest imaging. Please conduct formal statistical testing for interaction between trace-positive status and CXR findings. It may be helpful to present stratified hazard ratios and cumulative incidence curves for trace-positive participants with normal versus abnormal CXR. 

    Minor issues:

    1. Consider adding statistical significance indicators to Figure 4. It is difficult for the reader to interpret which differences between groups are statistically meaningful after Bonferroni correction.

    2. The manuscript emphasizes that "a CAD score threshold of 0.5 resulted in a sensitivity of 75% and a specificity of 64%, and a threshold of 0.2 had a sensitivity of 86% and specificity of 45%." Figure 5 could be strengthened by visually denoting the specific threshold points (0.5 and 0.2) that are discussed in the text. 

    Discussion

    Minor issues:

    1. The study's interpretation of "incident tuberculosis" would benefit from a more nuanced discussion of whether these cases represent true progression of early disease or delayed detection of prevalent tuberculosis that was below detection thresholds at baseline. While the authors frame trace results as potentially representing early tuberculosis that could progress over time, the study design cannot definitively distinguish between early TB that progresses, missed prevalent TB that was below the detection threshold, and reinfection during follow-up. Without molecular methods to confirm whether detected TB progression represents the same infection as detected by the initial trace result, alternative explanations for the observed "incident" cases remain plausible. Acknowledge the limitations in distinguishing true progression from delayed detection of prevalent disease.

    Competing interests

    The authors declare that they have no competing interests.

    Use of Artificial Intelligence (AI)

    The authors declare that they used generative AI to come up with new ideas for their review.

  2. Rapid Reviews Infectious Diseases

    Marisa Nielsen

    Review 3: "The Long-Term Risk of Tuberculosis among Individuals with Xpert Ultra "trace" Screening Results: A Longitudinal Follow-Up Study"

    Overall, this research strengthens the case for individualized, context-aware treatment decisions in TB control policies.

  3. Rapid Reviews Infectious Diseases

    Patrick Howlett

    Review 2: "The Long-Term Risk of Tuberculosis among Individuals with Xpert Ultra "trace" Screening Results: A Longitudinal Follow-Up Study"

    Overall, this research strengthens the case for individualized, context-aware treatment decisions in TB control policies.

  4. Rapid Reviews Infectious Diseases

    Syed Mohammed Rahman

    Review 1: "The Long-Term Risk of Tuberculosis among Individuals with Xpert Ultra "trace" Screening Results: A Longitudinal Follow-Up Study"

    Overall, this research strengthens the case for individualized, context-aware treatment decisions in TB control policies.

  6. Version published to 10.1101/2025.03.20.25324205v1 on medRxiv