Improved Diagnosis and Treatment Monitoring of Tuberculosis Using Stool and the Tuberculosis Bacterial Load Assay (TB-MBLA).

The diagnosis and monitoring of tuberculosis treatment is difficult as many patients are unable to produce sputum. This means that many patients are treated on the basis of clinical findings and consequently some will be exposed to anti-tuberculosis drugs unnecessarily. Moreover, for those appropriately on treatment and unable to produce a sputum sample, it will be impossible to monitor the response to treatment. We have shown that stool is a potential alternative sample type for diagnosis of tuberculosis. Currently, available protocols like the Xpert MTB/RIF use DNA as a target to detect Mycobacterium tuberculosis in stool but DNA survives long after the organism is dead so it is not certain whether a positive test is from an old or a partially treated infection. The TB MBLA only detects live organisms and thus, can be used to follow the response to treatment. In this chapter, we describe a protocol for TB-MBLA, an RNA-based assay, and apply it to quantify TB bacteria in stool.

Accuracy of the tuberculosis molecular bacterial load assay to diagnose and monitor response to anti-tuberculosis therapy: a longitudinal comparative study with standard-of-care smear microscopy, Xpert MTB/RIF Ultra, and culture in Uganda.

BACKGROUND: In 2018, the tuberculosis molecular bacterial load assay (TB-MBLA), a ribosomal RNA-based test, was acknowledged by WHO as a molecular assay that could replace smear microscopy and culture for monitoring tuberculosis treatment response. In this study, we evaluated the accuracy of TB-MBLA for diagnosis and monitoring of treatment response in comparison with standard-of-care tests. METHODS: For this longitudinal prospective study, patients aged 18 years or older with presumptive tuberculosis (coughing for at least 2 weeks, night sweats, and weight loss) were enrolled at China-Uganda Friendship Hospital Naguru (Kampala, Uganda). Participants were evaluated for tuberculosis by TB-MBLA in comparison with Xpert MTB/RIF Ultra (Xpert-Ultra) and smear microscopy, with Mycobacteria Growth Indicator Tube (MGIT) culture as a reference test. Participants who were positive on Xpert-Ultra were enrolled on a standard 6-month anti-tuberculosis regimen, and monitored for treatment response at weeks 2, 8, 17, and 26 after initiation of treatment and then 3 months after treatment. FINDINGS: Between Nov 15, 2019, and June 15, 2022, 210 participants (median age 35 years [IQR 27-44]) were enrolled. 135 (64%) participants were male and 72 (34%) were HIV positive. The pretreatment diagnostic sensitivities of TB-MBLA and Xpert-Ultra were similar (both 99% [95% CI 95-100]) but the specificity was higher for TB-MBLA (90% [83-96]) than for Xpert-Ultra (78% [68-86]). Ten participants were Xpert-Ultra trace positive, eight (80%) of whom were negative by TB-MBLA and MGIT culture. Smear microscopy had lower diagnostic sensitivity (75% [65-83]) but higher specificity (98% [93-100]) than TB-MBLA and Xpert-Ultra. Among participants who were smear microscopy negative, the sensitivity of TB-MBLA was 96% (95 CI 80-100) and was 100% (95% CI 86-100) in those who were HIV positive. 129 (61%) participants were identified as tuberculosis positive by Xpert-Ultra and these individuals were enrolled in the treatment group and monitored for treatment response. According to TB-MBLA, 19 of these patients cleared bacillary load to zero by week 2 of treatment and remained negative throughout the 6-month treatment follow-up. Positivity for tuberculosis decreased with treatment as measured by all tests, but the rate was slower with Xpert-Ultra. Consequently, 31 (33%) of 95 participants were still Xpert-Ultra positive at the end of treatment but were clinically well and negative on TB-MBLA and culture at 6 months of treatment. Two patients were still Xpert-Ultra positive with a further 3 months of post-treatment follow-up. The rate of conversion to negative of the DNA-based Xpert-Ultra was 3·3-times slower than that of the rRNA-based TB-MBLA. Consequently for the same patient, it would take 13 weeks and 52 weeks to reach complete tuberculosis negativity by TB-MBLA and Xpert-Ultra, respectively. Participants who were positive on smear microscopy at 8 weeks, who received an extra month of intensive treatment, had a similar TB-MBLA-measured bacillary load at 8 weeks to those who were smear microscopy negative. INTERPRETATION: TB-MBLA has a similar performance to Xpert-Ultra for pretreatment diagnosis of tuberculosis, but is more accurate at detecting and characterising the response to treatment than Xpert-Ultra and standard-of-care smear microscopy. FUNDING: European and Developing Countries Clinical Trials Partnership, Makerere University Research and Innovation Fund, US National Institutes of Health.

Diagnostic accuracy of Xpert MTB/RIF Ultra and culture assays to detect Mycobacterium Tuberculosis using OMNIgene-sputum processed stool among adult TB presumptive patients in Uganda.

BACKGROUND: Stool is a potential sample for diagnosing Mycobacterium tuberculosis (Mtb) in patients with difficulty in expectorating. However, high mycobacterial culture contamination rates and Xpert MTB/RIF Ultra test error rates on stool samples have limited its use. OMNIgene SPUTUM (OM-S) is a sample transport reagent with characteristics of sputum decontamination while maintaining viable Mtb. We evaluated the impact of OM-S on Mtb diagnostic yield from stool using smear microscopy, Xpert MTB/RIF Ultra, and culture among presumptive TB patients. METHODS: Paired stool and expectorated sputum samples were collected from consecutive Ugandan adults undergoing diagnostic evaluation for pulmonary TB between June 2018 and June 2019. Stool was divided into 2 portions: one was homogenized in OM-S (OM-S stool) and the other in PBS (PBS stool) as control. Both sputum and processed stool were tested for Mtb using concentrated smear fluorescence microscopy (CFM), Xpert MTB/RIF Ultra (Xpert) and Mycobacteria Growth Indicator Tube (MGIT) culture. Sensitivity, specificity, and predictive values for each test were calculated against sputum MGIT culture as the reference standard. RESULTS: Of the 200 participants, 120 (60%) were male, 73 (37%) were HIV positive (median CD4 120 cells/uL (IQR 43-297)) and 128 (64%) had confirmed pulmonary TB by sputum MGIT culture. Seven (4%) OM-S stool Xpert samples reported errors while 47 (25%) and 103 (61%) were contaminated on OM-S stool MGIT and PBS stool MGIT, respectively. OM-S stool MGIT was able to accurately diagnose 56 of the contaminated PBS stool MGIT samples compared to only 5 of the contaminated OM-S stool MGIT samples diagnosed by PBS stool MGIT. Sensitivity (95% Confidence Interval, CI) 89% (83-94) for OM-S stool Xpert was higher compared to that of OM-S stool MGIT 60% (51-69) and PBS stool MGIT 42% (32-52). Specificity (95%CI) 91% (82-97) was also higher for OM-S stool Xpert compared to OM-S stool MGIT 64% (51-75) and PBS stool MGIT 26% (16-38). CONCLUSION: Stool processed with OM-S showed potential to improve Mtb diagnostic yield and reduce rates of indeterminate results when tested on Xpert and MGIT culture. The method may thus be of value in Mtb detection among patients with difficulty to expectorate.

HIV infection is associated with elevated biomarkers of immune activation in Ugandan adults with pneumonia.

INTRODUCTION: Pneumonia is an important cause of morbidity and mortality in persons living with human immunodeficiency virus (HIV) infection. How immune activation differs among HIV-infected and HIV-uninfected adults with pneumonia is unknown. METHODS: The Inflammation, Aging, Microbes, and Obstructive Lung Disease (I AM OLD) Cohort is a prospective cohort of adults with pneumonia in Uganda. In this cross-sectional analysis, plasma was collected at pneumonia presentation to measure the following 12 biomarkers: interleukin 6 (IL-6), soluble tumor necrosis factor receptors 1 and 2 (sTNFR-1 and sTNFR-2), high sensitivity C-reactive protein (hsCRP), fibrinogen, D-dimer, soluble CD27 (sCD27), interferon gamma-inducible protein 10 (IP-10), soluble CD14 (sCD14), soluble CD163 (sCD163), hyaluronan, and intestinal fatty acid binding protein. We asked whether biomarker levels differed between HIV-infected and HIV-uninfected participants, and whether higher levels of these biomarkers were associated with mortality. RESULTS: One hundred seventy-three participants were enrolled. Fifty-three percent were HIV-infected. Eight plasma biomarkers-sTNFR-1, sTNFR-2, hsCRP, D-dimer, sCD27, IP-10, sCD14, and hyaluronan-were higher among participants with HIV infection, after adjustment for pneumonia severity. Higher levels of 8 biomarkers-IL-6, sTNFR-1, sTNFR-2, hsCRP, IP-10, sCD14, sCD163, and hyaluronan-were associated with increased 2-month mortality. CONCLUSIONS: As in other clinical contexts, HIV infection is associated with a greater degree of immune activation among Ugandan adults with pneumonia. Some of these are also associated with short-term mortality. Further study is needed to explore whether these biomarkers might predict poor long-term outcomes-such as the development of obstructive lung disease-in patients with HIV who have recovered from pneumonia.