Multiple pathogens contribute to human immunodeficiency virus-related sepsis in addition to Mycobacterium tuberculosis: A prospective cohort in Tanzania.

Background: Mortality from tuberculosis (TB) sepsis is common among patients living with human immunodeficiency virus (PLHIV). We aimed to detect M. tuberculosis (MTB) and additional sepsis etiologies, and mortality determinants in PLHIV. Methods: This prospective cohort study consented and followed-up PLHIV for 28 days in northern Tanzania. From May through December 2021, patients provided urine and sputum for TB testing in lateral-flow lipoarabinomannan (LF-LAM) and Xpert® MTB/RIF. Bacterial blood culture, cryptococcal antigen, malaria rapid diagnostic, C-reactive-protein (CRP), and international normalized ratio (INR) tests were also performed. Sepsis severity was clinically measured by Karnofsky and modified early warning signs (MEWS) scores. Anti-TB, broad-spectrum antibiotics, and antimalarial and antifungal agents were prescribed in accordance with Tanzania treatment guideline. An independent t-test and Chi-square or Fisher's exact tests compared means and proportions, respectively. P < 0.05 was statistically significant. Results: Among 98 patients, 59 (60.2%) were female. Their mean (standard deviation) age was 44 (12.9) years. TB detection increased from 24 (24.5%) by Xpert® MTB/RIF to 36 (36.7%) when LF-LAM was added. In total, 23 (23.5%) patients had other than TB etiologies of sepsis, including Staphylococcus aureus, Streptococcus pneumoniae, Cryptococcus spp., and Plasmodium spp. Twenty-four (94.4%) of 36 patients with TB had higher CRP (≥10 mg/l) compared to 25 (40.3%) non-TB patients (P < 0.001). Nine (9.2%) patients died and almost all had INR ≥1.8 (n = 8), Karnofsky score <50% (n = 9), MEWS score >6 (n = 8), and malnutrition (n = 9). Conclusions: MTB and other microbes contributed to sepsis in PLHIV. Adding non-TB tests informed clinical decisions. Mortality was predicted by conventional sepsis and severity scoring, malnutrition, and elevated INR.

Clinical-demographic markers for improving diabetes mellitus diagnosis in people with tuberculosis in Tanzania.

BACKGROUND: Tuberculosis (TB) control is threatened by an increasing prevalence of diabetes mellitus (DM), particularly in endemic countries. Screening for DM is not routinely implemented in Tanzania; therefore, we aimed to screen for DM at TB diagnosis using clinical-demographic markers. METHODS: Our cross-sectional study recruited TB patients who received anti-TB treatment between October 2019 and September 2020 at health care facilities in three regions from Tanzania. Patients were screened for DM using DM symptoms (polydipsia, polyphagia and polyuria) and random blood glucose (RBG) testing. Patients with a history of DM and those with no history of DM but an RBG ≥ 7.8 mmol/L had point-of-care glycated haemoglobin (HbA1c) testing, and were considered to have DM if HbA1c was ≥ 48 mmol/mol. RESULTS: Of 1344 TB patients, the mean age was 41.0 (± 17.0) years, and 64.7% were male. A total of 1011 (75.2%) had pulmonary TB, and 133 (10.4%) had at least one DM symptom. Overall, the prevalence of DM was 7.8%, of which 36 (2.8%) TB patients with no history of DM were newly diagnosed with DM by RBG testing. TB/DM patients were older than those with only TB (50.0 ± 14.0 years vs 40.0 ± 17.0 years, p < 0.001). Patients with RBG ≥ 7.8 mmol/L were more likely to have pulmonary TB (p = 0.003), age ≥ 35 years (p = 0.018), and have at least one DM symptom (p < 0.001). There was a substantial agreement (Kappa = 0.74) between the on-site glucometer and point-of-care HbA1c tests in detecting DM range of hyperglycemia. CONCLUSION: The implementation of clinical-demographic markers and blood glucose screening identified the overall prevalence of DM and those at risk of DM in TB patients. Clinical-demographic markers are independent predictors for DM range hyperglycemia and highlight the importance of further diagnostic testing and early co-management of TB and DM.

Blood cytokine responses to early secreted protein antigen-6/culture filtrate protein-10 tuberculosis antigens 2 months after antituberculosis treatment among patients with drug-susceptible pulmonary tuberculosis.

BACKGROUND: Human tuberculosis is a chronic inflammatory disease caused by mycobacterium tuberculosis. Pulmonary tuberculosis is the result of the failure of host immune system to control mycobacterium tuberculosis. The aim of the study was to asses the changes of the cytokines in active pulmonary tuberculosis patients before and after the use of anti-TB therapy. METHODS: Multiple cytokine responses in active tuberculosis (TB) patients were investigated in this study following anti-TB drug therapy after 2 months. Ninety-six participants with pulmonary TB were engaged in the study between May 2018 and October 2018. Samples of blood were taken early before treatment at 0 and 2 months after using anti-TB therapy. The levels of interferon-gamma (IFN)-γ, interleukin-4 (IL-4), IL-6, IL-10, and tumor necrosis factor (TNF)-α in whole blood plasma collected from the QuantiFERON-TB Gold Plus were measured. RESULTS: Compared with baseline levels, TNF-α, IL6 and IL10 were significantly lower following treatment whereas the IFN-γ and IL-4 increased significantly after treatment. The responses of five cytokines varied significantly after treatment (P < 0.0001) where IFN-γ was highest compared to other cytokines with 123.6%, AUC=0.757 and P < 0001, TNF-α AUC: 0.529 and P = 0.743, IL-4 AUC:0.557 and P = 0.514, IL-6 AUC:0.629 and P = 0.047, IL-10 AUC:0.549 and P = 0.581. CONCLUSION: It is concluded that changes of cytokines that observed during the treatment of TB patients play a very important role in monitoring pulmonary TB and can be suitable biomarkers to assess the effectiveness of anti-TB therapy in patients with TB.

Evaluation of cytokines in peripheral blood mononuclear cell supernatants for the diagnosis of tuberculosis.

INTRODUCTION: There is active interest in leveraging host immune responses as biomarkers of tuberculosis (TB) disease activity. We had previously evaluated an immunodiagnostic test called the antibody in lymphocyte supernatant (ALS) assay. Here, we aimed to evaluate a panel of inflammatory mediators and associate the responses with the ALS results to identify a biosignature to distinguish TB cases from controls. METHODOLOGY: In this case-control study, adults with TB were compared to controls who were hospitalized for non-infectious conditions. Blood was collected at baseline and after 4 weeks of TB treatment (from TB cases only). Peripheral blood mononuclear cells were isolated and cultured without antigenic stimulation for 72 hours. Inflammatory mediators were measured using the Multiplex cytokine kit and compared between TB cases and controls; among TB cases, responses were compared over time. ALS and inflammatory mediator results were evaluated using generalized discriminant analysis to identify the optimal biosignature to predict TB. RESULTS: When comparing inflammatory mediators between groups, IL-1ra, IL-1ß, and granulocyte macrophage-colony stimulating factor (GM-CSF) were lower in TB cases (P<0.002). Fibroblast growth factor-basic significantly increased from baseline to week-4 (P=0.002). Generalized discriminant analysis yielded a model with IL-2, tumor necrosis factor-alpha, vascular endothelial growth factor, and ALS, providing a sensitivity of 82.2% and specificity of 76.2%. CONCLUSION: Our results suggest that IL-1ra, IL-1ß, and GM-CSF might be used as diagnostic biomarkers to distinguish between TB cases and non-TB cases. We could not identify a group of mediators that outperformed the diagnostic accuracy of the ALS alone.

Phenotypic Changes on Mycobacterium Tuberculosis-Specific CD4 T Cells as Surrogate Markers for Tuberculosis Treatment Efficacy.

Background: The analysis of phenotypic characteristics on Mycobacterium tuberculosis (MTB)-specific T cells is a promising approach for the diagnosis of active tuberculosis (aTB) and for monitoring treatment success. We therefore studied phenotypic changes on MTB-specific CD4 T cells upon anti-tuberculosis treatment initiation in relation to the treatment response as determined by sputum culture. Methods: Peripheral blood mononuclear cells from subjects with latent MTB infection (n = 16) and aTB (n = 39) at baseline, weeks 9, 12, and 26 (end of treatment) were analyzed after intracellular interferon gamma staining and overnight stimulation with tuberculin. Liquid sputum cultures were performed weekly until week 12 and during 4 visits until week 26. Results: T cell activation marker expression on MTB-specific CD4 T cells differed significantly between subjects with aTB and latent MTB infection with no overlap for the frequencies of CD38pos and Ki67pos cells (both p < 0.0001). At 9 weeks after anti-TB treatment initiation the frequencies of activation marker (CD38, HLA-DR, Ki67) positive MTB-specific, but not total CD4 T cells, were significantly reduced (p < 0.0001). Treatment induced phenotypic changes from baseline until week 9 and until week 12 differed substantially between individual aTB patients and correlated with an individual's time to stable sputum culture conversion for expression of CD38 and HLA-DR (both p < 0.05). In contrast, the frequencies of maturation marker CD27 positive MTB-specific CD4 T cells remained largely unchanged until week 26 and significantly differed between subjects with treated TB disease and latent MTB infection (p = 0.0003). Discussion: Phenotypic changes of MTB-specific T cells are potential surrogate markers for tuberculosis treatment efficacy and can help to discriminate between aTB (profile: CD38pos, CD27low), treated TB (CD38neg, CD27low), and latent MTB infection (CD38neg, CD27high).

Platelet-monocyte interaction in Mycobacterium tuberculosis infection.

The immune effects of platelets and platelet-leukocyte aggregation are increasingly recognized. We studied the occurrence of platelet-monocyte aggregation (PMA) in patients with pulmonary tuberculosis (TB), the processes underlying PMA and consequences for cytokine responses. In a cross-sectional study involving 65 Tanzanian TB patients in different phases of treatment and 29 healthy controls, TB patients had a significantly higher PMA. This increased PMA in TB patients was associated with increased monocyte CCR5, CD16 expression and PF4, but not with increased membrane-expressed or soluble P-selectin expression. These findings were confirmed in vitro: whereas incubation of whole blood with Mycobacterium tuberculosis (Mtb) did not activate platelets, monocytes became activated with higher CD11b, CD16 and CCR5 expression, but this was independent of platelet-monocyte interaction. Still, platelets had an anti-inflammatory effect on cytokine responses as peripheral blood mononuclear cells (PBMC) incubated with Mtb in the presence of platelets produced less interleukin (IL)-1ß, tumor necrosis factor-α, IL-6 and interferon-γ and more IL-10. In conclusion, increased PMA during TB infection is caused by monocyte and not platelet activation. By counteracting the Mtb-induced pro-inflammatory leukocyte response, platelets may protect against excessive tissue damage, but may also compromise the production of protective cytokines, such as IFNÆ´ and TNFα.

Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6.

There is a need for an improved vaccine for tuberculosis. ESAT-6 is a cardinal vaccine antigen with unique properties and is included in several vaccine candidates in development. ESAT-6 is also the core antigen in the IFN-γ release assays (IGRA) used to diagnose latent infection, rendering IGRA tests unspecific after vaccination. This challenge has prompted the development of a companion diagnostic for ESAT-6 based vaccines, an ESAT-6 free IGRA. We screened a panel of seven potential new diagnostic antigens not recognized in BCG vaccinated individuals. Three highly recognized antigens EspC, EspF and Rv2348c were identified and combined with CFP10 in an ESAT-6 free antigen cocktail. The cocktail was prepared in a field-friendly format, lyophilized with heparin in ready-to-use vacutainer tubes. The diagnostic performance of the ESAT-6 free IGRA was determined in a cross-validation study. Compared IGRA, the ESAT-6 free IGRA induced a comparable magnitude of IFN-γ release, and the diagnostic performance was on par with Quantiferon (sensitivity 84% vs 79%; specificity 99% vs 97%). The comparable performance of the ESAT-6 free IGRA to IGRA suggests potential as companion diagnostic for ESAT-6 containing vaccines and as adjunct test for latent infection.

Glycated hemoglobin screening identifies patients admitted for retreatment of tuberculosis at risk for diabetes in Tanzania.

INTRODUCTION: World Health Organization recommendations of bidirectional screening for tuberculosis (TB) and diabetes have been met with varying levels of uptake by national TB programs in resource-limited settings. METHODOLOGY: Kibong'oto Infectious Diseases Hospital (KIDH) is a referral hospital for TB from northern Tanzania, and the national referral hospital for multidrug-resistant (MDR)-TB. Glycated hemoglobin (HgbA1c) testing was done on patients admitted to KIDH for newly diagnosed TB, retreatment TB, and MDR-TB, to determine the point prevalence of diabetes (HgbA1c ≥ 6.5%) and prediabetes (HgbA1c 5.7%-6.4%). RESULTS: Of 148 patients hospitalized at KIDH over a single week, 59 (38%) had no prior TB treatment, 22 (15%) were retreatment cases, and 69 (47%) had MDR-TB. Only 3 (2%) had a known history of diabetes. A total of 144 (97%) had successful screening, of which 110 (77%) had an HgbA1c ≤ 5.6%, 28 (19%) had ≥ 5.7 < 6.5, and 6 (4%) had ≥ 6.5. Comparing subjects with prediabetes or diabetes to those with normal A1c levels, retreatment patients were significantly more likely to have a A1c ≥ 5.7% (odds ratio: 3.2, 95% CI: 1.2-9.0; p = 0.02) compared to those without prior TB treatment. No retreatment case was a known diabetic, thus the number needed to screen to diagnose one new case of diabetes among retreatment cases was 11. CONCLUSIONS: Diabetes prevalence by HgbA1c was less common than expected, but higher HgA1c values were significantly more frequent among retreatment cases, allowing for a rational, resource-conscious screening approach.

Diagnosis and interim treatment outcomes from the first cohort of multidrug-resistant tuberculosis patients in Tanzania.

SETTING: Kibong'oto National Tuberculosis Hospital (KNTH), Kilimanjaro, Tanzania. OBJECTIVE: Characterize the diagnostic process and interim treatment outcomes from patients treated for multidrug-resistant tuberculosis (MDR-TB) in Tanzania. DESIGN: A retrospective cohort study was performed among all patients treated at KNTH for pulmonary MDR-TB between November 2009 and September 2011. RESULTS: Sixty-one culture-positive MDR-TB patients initiated therapy, 60 (98%) with a prior history of TB treatment. Forty-one (67%) were male and 9 (14%) were HIV infected with a mean CD4 count of 424 (±106) cells/µl. The median time from specimen collection to MDR-TB diagnosis and from diagnosis to initiation of MDR-TB treatment was 138 days (IQR 101-159) and 131 days (IQR 32-233), respectively. Following treatment initiation four (7%) patients died (all HIV negative), 3 (5%) defaulted, and the remaining 54 (89%) completed the intensive phase. Most adverse drug reactions were mild to moderate and did not require discontinuation of treatment. Median time to culture conversion was 2 months (IQR 1-3) and did not vary by HIV status. In 28 isolates available for additional second-line drug susceptibility testing, fluoroquinolone, aminoglycoside and para-aminosalicylic acid resistance was rare yet ethionamide resistance was present in 9 (32%). CONCLUSION: The majority of MDR-TB patients from this cohort had survived a prolonged referral process, had multiple episodes of prior TB treatment, but did not have advanced AIDS and converted to culture negative early while completing an intensive inpatient regimen without serious adverse event. Further study is required to determine the clinical impact of second-line drug susceptibility testing and the feasibility of alternatives to prolonged hospitalization.