Novel Automation of an Enzyme-Linked Immunosorbent Spot Assay Testing Method: Comparable Diagnostic Performance of the T-SPOT.TB Test Using Manual Density Gradient Cell Isolation versus Automated Positive Selection with the T-Cell Select Kit.

The diagnosis of latent tuberculosis (TB) infection (LTBI) is critical to improve TB treatment and control, and the T-SPOT.TB test is a commercial enzyme-linked immunosorbent spot assay used for this purpose. The objective of the study was to increase automation and extend the time between blood collection and processing for the T-SPOT.TB test from 0 to 8 h to 0 to 54 h. The previous maximum time between blood collection and processing for the T-SPOT.TB test is 32 h using T-Cell Xtend. For this, we compared the T-SPOT.TB test using manual peripheral blood mononuclear cell (PBMC) isolation by density gradient separation at 0 to 8 h (reference method, control arm) to an automated PBMC isolation method using magnetic beads (T-Cell Select kit) at 0 to 55 h postcollection. A total of 620 subjects were enrolled from 4 study sites, and blood samples were collected from each volunteer, comprising 1,850 paired samples in total. Overall agreement between both methods was 96.8% (confidence interval [CI], 95.9 to 97.6%), with 95.8% (CI, 93.5 to 97.5%) positive and 97.1% negative agreement (CI, 96.1 to 97.9%). In summary, there was a strong overall agreement between the automated and manual T-SPOT.TB test processing methods. The results suggest that the T-SPOT.TB test can be processed using automated positive selection with magnetic beads using T-Cell Select to decrease hands-on time. Also, this cell isolation method allowed for the time between blood collection and processing to range from 0 to 55 h. Additional studies in larger and diverse patient populations including immunocompromised and pediatric patients are needed.

Antigen specificity shapes antibody functions in tuberculosis.

Tuberculosis (TB) is the number one infectious disease cause of death worldwide due to an incomplete understanding of immunity. Emerging data highlight antibody functions mediated by the Fc domain as immune correlates. However, the mechanisms by which antibody functions impact the causative agent Mycobacterium tuberculosis (Mtb) are unclear. Here, we examine how antigen specificity determined by the Fab domain shapes Fc effector functions against Mtb. Using the critical structural and secreted virulence proteins Mtb cell wall and ESAT-6 & CFP-10, we observe that antigen specificity alters subclass, antibody post-translational glycosylation, and Fc effector functions in TB patients. Moreover, Mtb cell wall IgG3 enhances disease through opsonophagocytosis of extracellular Mtb . In contrast, polyclonal and a human monoclonal IgG1 we generated targeting ESAT-6 & CFP-10 inhibit intracellular Mtb . These data show that antibodies have multiple roles in TB and antigen specificity is a critical determinant of the protective and pathogenic capacity.

Interaction between type 2 diabetes and past COVID-19 on active tuberculosis.

BACKGROUND: The global setback in tuberculosis (TB) prevalence and mortality in the post-COVID-19 era have been partially attributed to pandemic-related disruptions in healthcare systems. The additional biological contribution of COVID-19 to TB is less clear. The goal of this study was to determine if there is an association between COVID-19 in the past 18 months and a new TB episode, and the role played by type 2 diabetes mellitus (DM) comorbidity in this relationship. METHODS: A cross-sectional study was conducted among 112 new active TB patients and 373 non-TB controls, identified between June 2020 and November 2021 in communities along the Mexican border with Texas. Past COVID-19 was based on self-report or positive serology. Bivariable/multivariable analysis were used to evaluate the odds of new TB in hosts with past COVID-19 and/or DM status. RESULTS: The odds of new TB were higher among past COVID-19 cases vs. controls, but only significant among DM patients (aOR 2.3). The odds of TB given DM was 2.7-fold among participants without past COVID-19 and increased to 7.9-fold among those with past COVID-19. CONCLUSION: DM interacts with past COVID-19 synergistically to magnify the risk of TB. Latent TB screening and prophylactic treatment, if positive, is recommended in this COVID-19/DM/latent TB high-risk group.

Differential Role of Type 2 Diabetes as a Risk Factor for Tuberculosis in the Elderly versus Younger Adults.

The elderly are understudied despite their high risk of tuberculosis (TB). We sought to identify factors underlying the lack of an association between TB and type 2 diabetes (T2D) in the elderly, but not adults. We conducted a case-control study in elderly (≥65 years old; ELD) vs. younger adults (young/middle-aged adults (18-44/45-64 years old; YA|MAA) stratified by TB and T2D, using a research study population (n = 1160) and TB surveillance data (n = 8783). In the research study population the adjusted odds ratio (AOR) of TB in T2D was highest in young adults (AOR 6.48) but waned with age becoming non-significant in the elderly. Findings were validated using TB surveillance data. T2D in the elderly (vs. T2D in younger individuals) was characterized by better glucose control (e.g., lower hyperglycemia or HbA1c), lower insulin resistance, more sulphonylureas use, and features of less inflammation (e.g., lower obesity, neutrophils, platelets, anti-inflammatory use). We posit that differences underlying glucose dysregulation and inflammation in elderly vs. younger adults with T2D, contribute to their differential association with TB. Studies in the elderly provide valuable insights into TB-T2D pathogenesis, e.g., here we identified insulin resistance as a novel candidate mechanism by which T2D may increase active TB risk.

Human monocyte-derived macrophage responses to M. tuberculosis differ by the host's tuberculosis, diabetes or obesity status, and are enhanced by rapamycin.

Human macrophages play a major role in controlling tuberculosis (TB), but their anti-mycobacterial mechanisms remain unclear among individuals with metabolic alterations like obesity (TB protective) or diabetes (TB risk). To help discern this, we aimed to: i) Evaluate the impact of the host's TB status or their comorbidities on the anti-mycobacterial responses of their monocyte-derived macrophages (MDMs), and ii) determine if the autophagy inducer rapamycin, can enhance these responses. We used MDMs from newly diagnosed TB patients, their close contacts and unexposed controls. The MDMs from TB patients had a reduced capacity to activate T cells (surrogate for antigen presentation) or kill M. tuberculosis (Mtb) when compared to non-TB controls. The MDMs from obese participants had a higher antigen presenting capacity, whereas those from chronic diabetes patients displayed lower Mtb killing. The activation of MDMs with rapamycin led to an enhanced anti-mycobacterial activity irrespective of TB status but was not as effective in patients with diabetes. Further studies are warranted using MDMs from TB patients with or without metabolic comorbidities to: i) elucidate the mechanisms through which host factors affect Mtb responses, and ii) evaluate host directed therapy using autophagy-inducing drugs like rapamycin to enhance macrophage function.

COVID-19 and chronic diabetes: the perfect storm for reactivation tuberculosis?: a case series.

BACKGROUND: The coronavirus disease 2019 pandemic is predicted to have a net negative effect on tuberculosis control, with an estimated excess of 6.3 million tuberculosis cases and 1.4 million deaths by 2025. Programmatic issues such as the lockdown of tuberculosis services affect all patients, while biosocial factors have a differential impact on an individual's risk for tuberculosis or adverse tuberculosis outcomes. CASE PRESENTATION: We report three Hispanic cases of incident tuberculosis (two males, 43 and 44 years old; one female, 49 years old) after resolution of coronavirus disease episodes. Coincidentally, all cases shared a common risk factor: a chronic history poorly controlled diabetes. CONCLUSIONS: Our findings alert to the threat posed by the synergy between coronavirus disease and diabetes, on tuberculosis reactivation. In medium- to high-risk settings for tuberculosis, we recommend implementation of routine screening for latent tuberculosis infection in these cases, and preventive tuberculosis treatment in those who are positive.

Interferon gamma release assays for detection of latent Mycobacterium tuberculosis in older Hispanic people.

BACKGROUND: Interferon gamma release assays (IGRAs) are used to detect latent Mycobacterium tuberculosis (M.tb) infection (LTBI) in adults, but their performance in older people is not well-established. We evaluated IGRAs for LTBI detection in older Hispanic recent TB contacts (ReC) or community controls (CoC). METHODS: Cross-sectional assessment of LTBI with T-SPOT.TB and/or QuantiFERON-Gold in-tube or -Plus assay in older (≥60 years) and adult (18-50 years) Hispanic people. RESULTS: We enrolled 193 CoC (119 adults, 74 older persons) and 459 ReC (361 adults, 98 older persons). LTBI positivity increased with age in CoC (19%-59%, P<0.001), but was similar in ReC (59%-69%, P=0.329). Older people had lower concordance between IGRAs (kappa 0.465 vs 0.688 in adults) and more inconclusive results (indeterminate/borderline 11.6% vs 5.8% in adults, P=0.012). With simultaneous IGRAs, inconclusive results were resolved as positive or negative with the other IGRA. The magnitude of response to M.tb peptides in IGRAs was similar among age groups, but responsiveness to mitogens was lower in older people. CONCLUSIONS: IGRAs are suitable for LTBI detection in older people. Discordant and inconclusive findings are more prevalent in older people, but results are resolved when IGRA is repeated with a different IGRA test.

A prospective cross-sectional study of tuberculosis in elderly Hispanics reveals that BCG vaccination at birth is protective whereas diabetes is not a risk factor.

BACKGROUND: Aging increases the risk of tuberculosis (TB) and its adverse outcomes, but most studies are based on secondary analyses, and few are in Hispanics. Diabetes is a risk factor for TB in adults, but its contribution in the elderly is unknown. We aimed to identify the role of diabetes and other risk factors for TB in elderly Hispanics. METHODS: Cross-sectional study among newly-diagnosed TB patients, recent contacts (ReC), or community controls (CoC) totaling 646 participants, including 183 elderly (>60 years; 43 TB, 80 ReC, 60 CoC) and 463 adults (18 to 50 years; 80 TB, 301 ReC and 82 CoC). Host characteristics associated with TB and latent Mycobacterium tuberculosis infection (LTBI) were identified in the elderly by univariable and confirmed by multivariable logistic regression. RESULTS: LTBI was more prevalent among the elderly CoC (55% vs. 23.2% in adults; p<0.001), but not in ReC (elderly 71.3% vs. adult 63.8%); p = 0.213). Risk factors for TB in the elderly included male sex (adj-OR 4.33, 95% CI 1.76, 10.65), smoking (adj-OR 2.55, 95% CI 1.01, 6.45) and low BMI (adj-OR 12.34, 95% CI 4.44, 34.33). Unexpectedly, type 2 diabetes was not associated with TB despite its high prevalence (adj-OR 0.38, 95% CI 0.06, 2.38), and BCG vaccination at birth was protective (adj-OR 0.16, 95% CI 0.06, 0.45). CONCLUSIONS: We report novel distinctions in TB risk factors in the elderly vs. adults, notably in diabetes and BCG vaccination at birth. Further studies are warranted to address disparities in this vulnerable, understudied population.

Diabetes screen during tuberculosis contact investigations highlights opportunity for new diabetes diagnosis and reveals metabolic differences between ethnic groups.

Type 2 diabetes (T2D) is a prevalent risk factor for tuberculosis (TB), but most studies on TB-T2D have focused on TB patients, been limited to one community, and shown a variable impact of T2D on TB risk or treatment outcomes. We conducted a cross-sectional assessment of sociodemographic and metabolic factors in adult TB contacts with T2D (versus no T2D), from the Texas-Mexico border to study Hispanics, and in Cape Town to study South African Coloured ethnicities. The prevalence of T2D was 30.2% in Texas-Mexico and 17.4% in South Africa, with new diagnosis in 34.4% and 43.9%, respectively. Contacts with T2D differed between ethnicities, with higher smoking, hormonal contraceptive use and cholesterol levels in South Africa, and higher obesity in Texas-Mexico (p < 0.05). PCA analysis revealed striking differences between ethnicities in the relationships between factors defining T2D and dyslipidemias. Our findings suggest that screening for new T2D in adult TB contacts is effective to identify new T2D patients at risk for TB. Furthermore, studies aimed at predicting individual TB risk in T2D patients, should take into account the heterogeneity in dyslipidemias that are likely to modify the estimates of TB risk or adverse treatment outcomes that are generally attributed to T2D alone.