Enhanced tuberculosis detection in advanced HIV using stool molecular testing

The Xpert MTB/Ultra molecular diagnostic test for stool samples, until now recommended only for children, could be established as an additional test for diagnosing tuberculosis in adults living with HIV. This is the main conclusion of the Stool4TB Alliance study, led by the Barcelona Institute for Global Health (ISGlobal), an institution supported by the "la Caixa" Foundation, in collaboration with the Manhiça Health Research Centre (CISM), the Research Center Borstel, the Makerere University, the Baylor College of Medicine Children's Foundation – Eswatini, and The Amsterdam Institute for Global Health and Development. The results, published in The Lancet Microbe, could represent a paradigm shift in the diagnosis of the disease in this population. 

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, was responsible for 1.25 million deaths in 2023, 13% of which were among people living with HIV. Currently, the main diagnostic strategy for tuberculosis focuses on sputum samples, which are obtained by deep coughing and expectoration of secretions produced in the lungs. So far, the WHO has recommended molecular sputum tests for people living with HIV, in addition to urine antigen detection (TB-LAM). However, access to these diagnostics remains limited in many settings, and they are not effective for all people living with HIV. In fact, people with HIV often struggle to produce sputum; more than half of those in advanced stages of the disease are unable to do so. In addition, the concentration of bacteria in the sputum is often so low that it is undetectable.

To address this problem, researchers from the EDCTP-funded Stool4TB project proposed using a molecular test (called Xpert MTB/RIF Ultra), currently used on respiratory samples, on stool samples. This test is already recommended for children because they have difficulty producing sputum. The research was conducted between December 2021 and August 2024 in medical centres in three African countries -Eswatini, Mozambique, and Uganda-, and recruited 677 patients over 15 years old with HIV and suspected TB. Participants provided sputum, urine, stool, and blood samples.

The challenge of diagnosing TB in vulnerable populations

"People living with HIV are at higher risk of developing pulmonary tuberculosis, but diagnosis in these cases is particularly challenging due to the low sensitivity of conventional tests," explains George William Kasule, a PhD student at ISGlobal and the University of Barcelona, and the first author of the study.

The variety of samples obtained in this study allowed us to compare the sensitivity and specificity of the Stool Ultra test with a microbiological reference standard consisting of three WHO-recommended tests: TB-LAM in urine, liquid culture, and Xpert Ultra from sputum."

George William Kasule, PhD Student, ISGlobal

A more sensitive test in advanced stages of HIV

The results showed that the stool test had a sensitivity of 23.7% and a specificity of 94.0%, compared with the reference standard. In patients with CD4 counts below 200 cells/μl, sensitivity increased to 45.5%. CD4 lymphocytes are immune system cells that are affected by HIV infection, so people with CD4 counts below 200 are more likely to develop serious opportunistic infections such as tuberculosis.

"The results of our study support the use of the Stool Ultra test as a complementary tool for diagnosing tuberculosis in people living with HIV, especially in those with advanced AIDS, where the risk of tuberculosis is higher," says Alberto L. García-Basteiro, a researcher at ISGlobal and head of the Vaccine and Immune Response to Infections Unit at Hospital Clínic de Barcelona.

The Stool Ultra test identified additional cases that were not detected by TB-LAM, Ultra in sputum or bacterial culture. "In these patients, the sensitivity of standard tests is much lower than in the HIV-negative population. However, in patients with advanced AIDS, our results show that stool molecular detection is no less effective than sputum testing. Most importantly, it can confirm the disease in many cases where respiratory tests are negative. This demonstrates the potential of this sample, perhaps counterintuitively, for diagnosing tuberculosis in people with HIV, especially when respiratory samples are not available," concludes García-Basteiro.