Ultrasensitive Assay Reveals Previously Undetected Tuberculosis in Hospital Patients

Tuberculosis remains the leading cause of death from an infectious disease worldwide, and diagnosis can be difficult when bacterial load is low or disease is atypical. In the United States, the disease killed nearly 600 people and sickened more than 9,600 in 2023, while elimination efforts have stalled with case counts rising since 2021. Conventional strategies often depend on culture of viable bacteria, which may fail to detect disease. New findings demonstrate that an ultrasensitive molecular assay can uncover previously undetected Mycobacterium tuberculosis DNA in hospitalized patients.

The Totally Optimized PCR (TOP) tuberculosis assay developed at Boston University is an ultrasensitive method for the direct detection of M. tuberculosis DNA in respiratory specimens. The assay targets a gene involved in bacterial cell-wall assembly and is designed to detect very low-level nucleic acid signals. According to investigators, this capability highlights limitations of diagnostic approaches that rely heavily on mycobacterial culture.

In three separate studies conducted over six years, researchers tested 297 respiratory samples from patients hospitalized at Boston Medical Center and St. Elizabeth’s Medical Center. The cohort was predominantly U.S.-born. Using the TOP assay, TB DNA was detected in 12–16% of respiratory samples despite Boston’s low tuberculosis incidence rate.

The results also showed that all three patients diagnosed during the study period with acute chest syndrome, a life‑threatening complication of sickle cell disease, were positive for TB DNA by TOP. Seventy‑five percent of TB DNA-positive patients were age 50 or older. Notably, most TB DNA-positive patients were negative on standard tuberculosis infection tests, including the tuberculin skin test and interferon‑gamma release assays.

The TOP assay was reported to be far more sensitive than standard mycobacterial cultures and other molecular tests in this series, and it has been validated in over 400 patients with suspected tuberculosis in Uganda, Brazil, and the United States. The study, published in Nature Communications, emphasizes the need for larger, prospective multicenter investigations with comprehensive clinical, radiological, immunological, and microbiological correlation. The authors contend that disseminating these preliminary signals is warranted given potential implications for medical care and public health.

“What we found was completely unexpected. Our ultrasensitive test is detecting M. tuberculosis DNA in patients who are unlikely to be diagnosed with TB using current methods,” said Guillermo Madico, scientist at Boston University’s National Emerging Infectious Diseases Laboratories (NEIDL) and co‑inventor of the TOP TB assay. "This opens the possibility that there could be thousands of Americans infected with forms of tuberculosis disease that remain hidden from our current diagnostic tools—putting them at risk of developing more serious complications or potentially transmitting the disease to others."

"These findings suggest we may be missing a significant burden of TB disease, particularly in older Americans and in patients with certain underlying conditions," said Dr. Jones-López, who conducted the research while at Boston Medical Center and Boston University Chobanian & Avedisian School of Medicine.  "Most concerning is the potential association with acute chest syndrome in sickle cell patients. If confirmed and expanded upon in larger studies, this finding could lead to better health outcomes for patients with this potentially life-threatening condition." 

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Boston University Chobanian & Avedisian School of Medicine.