Mycobacterium tuberculosis Lineage 7 Strains Are Associated with Prolonged Patient Delay in Seeking Treatment for Pulmonary Tuberculosis in Amhara Region,Ethiopia

Recent genotyping studies of Mycobacterium tuberculosis in Ethiopia have reported the identification of a new phylogenetically distinct M. tuberculosis lineage, lineage 7. We therefore investigated the genetic diversity and association of specific M. tuberculosis lineages with sociodemographic and clinical parameters among pulmonary TB patients in the Amhara Region, Ethiopia. DNA was isolated from M. tuberculosis-positive sputum specimens (n = 240) and analyzed by PCR and 24-locus mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) analysis and spoligotyping. Bioinformatic analysis assigned the M. tuberculosis genotypes to global lineages, and associations between patient characteristics and genotype were evaluated using logistic regression analysis. The study revealed a high diversity of modern and premodern M. tuberculosis lineages, among which approximately 25% were not previously reported. Among the M. tuberculosis strains (n = 138) assigned to seven subgroups, the largest cluster belonged to the lineage Central Asian (CAS) (n = 60; 26.0%), the second largest to lineage 7 (n = 36; 15.6%), and the third largest to the lineage Haarlem (n = 35; 15.2%). Four sublineages were new in the MIRU-VNTRplus database, designated NW-ETH3, NW-ETH1, NW-ETH2, and NW-ETH4, which included 24 (10.4%), 18 (7.8%), 8 (3.5%), and 5 (2.2%) isolates, respectively. Notably, patient delay in seeking treatment was significantly longer among patients infected with lineage 7 strains (Mann-Whitney test, P < 0.008) than in patients infected with CAS strains (adjusted odds ratio [AOR], 4.7; 95% confidence interval [CI], 1.6 to 13.5). Lineage 7 strains also grew more slowly than other M. tuberculosis strains. Cases of Haarlem (OR, 2.8; 95% CI, 1.2 to 6.6) and NW-ETH3 (OR, 2.8; 95% CI, 1.0 to 7.3) infection appeared in defined clusters. Intensified active case finding and contact tracing activities in the study region are needed to expedite diagnosis and treatment of TB.     

Peripheral blood gamma interferon release assays predict lung responses and Mycobacterium tuberculosis disease outcome in mice

Current diagnostic tests for tuberculosis (TB) are not able to distinguish active disease from latent Mycobacterium tuberculosis infection, nor are they able to quantify the risk of a latently infected person progressing to active TB. There is interest, however, in adapting antigen-specific gamma interferon (IFN-γ) release assays (IGRAs) to predict disease outcome. In this study, we used the differential susceptibilities of inbred mouse strains to M. tuberculosis infection to evaluate the prognostic capabilities of IGRAs. Using lung and blood cultures, we determined that CBA/J, DBA/2, and C3H/HeJ mice (models of heightened risk of progression to active TB) produced less antigen-specific IFN-γ in response to M. tuberculosis culture filtrate proteins and early secreted antigenic target-6 than the relatively resistant C57BL/6 mouse strain. Additionally, reduced IFN-γ secretion in supernatants reflected a reduced frequency of IFN-γ-responding cells in the lung and blood and not a specific defect in IFN-γ secretion at the single-cell level. Importantly, detection of antigen-specific IFN-γ from blood cultures accurately reflected lung responses, indicating that blood can be an appropriate test tissue in humans. Furthermore, reduced antigen-specific IFN-γ production and low frequencies of IFN-γ-responding cells from peripheral blood predicted increased risk of TB disease progression across genetically diverse TB disease-susceptible mouse strains, suggesting that similar results may occur in humans. The development of efficacious predictive diagnostic tests for humans would lead to targeted therapy prior to progression to active TB, reducing transmission, incidence, and prevalence rates while maximizing the use of public health resources.     

Mouse models of human TB pathology: roles in the analysis of necrosis and the development of host-directed therapies

A key aspect of TB pathogenesis that maintains Mycobacterium tuberculosis in the human population is the ability to cause necrosis in pulmonary lesions. As co-evolution shaped M . tuberculosis (M.tb) and human responses, the complete TB disease profile and lesion manifestation are not fully reproduced by any animal model. However, animal models are absolutely critical to understand how infection with virulent M.tb generates outcomes necessary for the pathogen transmission and evolutionary success. In humans, a wide spectrum of TB outcomes has been recognized based on clinical and epidemiological data. In mice, there is clear genetic basis for susceptibility. Although the spectra of human and mouse TB do not completely overlap, comparison of human TB with mouse lesions across genetically diverse strains firmly establishes points of convergence. By embracing the genetic heterogeneity of the mouse population, we gain tremendous advantage in the quest for suitable in vivo models. Below, we review genetically defined mouse models that recapitulate a key element of M.tb pathogenesis—induction of necrotic TB lesions in the lungs—and discuss how these models may reflect TB stratification and pathogenesis in humans. The approach ensures that roles that mouse models play in basic and translational TB research will continue to increase allowing researchers to address fundamental questions of TB pathogenesis and bacterial physiology in vivo using this well-defined, reproducible, and cost-efficient system. Combination of the new generation mouse models with advanced imaging technologies will also allow rapid and inexpensive assessment of experimental vaccines and therapies prior to testing in larger animals and clinical trials.     

Increased Severity of Tuberculosis in Guinea Pigs with Type 2 Diabetes: A Model of Diabetes-Tuberculosis Comorbidity

Impaired glucose tolerance and type 2 diabetes were induced in guinea pigs to model the emerging comorbidity of Mycobacterium tuberculosis infection in diabetic patients. Type 2 diabetes mellitus was induced by low-dose streptozotocin in guinea pigs rendered glucose intolerant by first feeding a high-fat, high-carbohydrate diet before M. tuberculosis exposure. M. tuberculosis infection of diabetic guinea pigs resulted in severe and rapidly progressive tuberculosis (TB) with a shortened survival interval, more severe pulmonary and extrapulmonary pathology, and a higher bacterial burden compared with glucose-intolerant and nondiabetic controls. Compared with nondiabetics, diabetic guinea pigs with TB had an exacerbated proinflammatory response with more severe granulocytic inflammation and higher gene expression for the cytokines/chemokines interferon-γ, IL-17A, IL-8, and IL-10 in the lung and for interferon-γ, tumor necrosis factor-α, IL-8, and monocyte chemoattractant protein-1 in the spleen. TB disease progression in guinea pigs with impaired glucose tolerance was similar to that of nondiabetic controls in the early stages of infection but was more severe by day 90. The guinea pig model of type 2 diabetes–TB comorbidity mimics important features of the naturally occurring disease in humans. This model will be beneficial in understanding the complex pathogenesis of TB in diabetic patients and to test new strategies to improve TB and diabetes control when the two diseases occur together.Host susceptibility to Mycobacterium tuberculosis is influenced by a variety of chronic communicable and noncommunicable diseases that increase the risk of infection and the development of active tuberculosis (TB) disease. Moreover, M. tuberculosis–infected patients with concurrent diseases often have more severe TB, which further complicates treatment responses to conventional antimicrobial drugs. The risk factors most frequently linked to M. tuberculosis susceptibility are HIV infection, malnutrition, tobacco use, air pollution, alcoholism, extremes in age, chronic kidney disease, and diabetes. The highest relative risk for TB is associated with profound immunosuppression associated with HIV infection. However, recent epidemiologic evidence suggests that in the face of a growing diabetes epidemic, the population-attributable risk of diabetes may be equivalent to or exceed that of HIV/AIDS. In countries with the highest TB and diabetes incidences, type 2 diabetes mellitus in particular may account for up to 20% of active TB cases, whereas <5% may be attributable to HIV.^1,2Type 2 diabetes accounts for approximately 95% of diabetes cases and is associated with obesity, poor diet, and a sedentary lifestyle, all of which are often linked to urbanization. An estimated 371 million people were diagnosed as having type 2 diabetes in 2012, with most residing in low- and middle-income countries. Moreover, an additional 280 million people are prediabetic, many of whom have undiagnosed insulin resistance with nondiabetic hyperglycemia and impaired glucose tolerance (IGT).³ Furthermore, the global incidence of type 2 diabetes is projected to rise to approximately 552 million by 2030.⁴ Evidence in human studies suggests that glucose control, more than any other feature of altered metabolism in diabetic patients, influences the susceptibility to M. tuberculosis infection, highlighting the importance of uncontrolled hyperglycemia in TB risk.⁵ The increased difficulty in controlling blood glucose levels in diabetic patients with TB, combined with poor responses to antimicrobial drug treatment, has the potential to further hamper current TB control efforts worldwide.⁶ An animal model that more closely mimics the pathogenesis of this comorbidity in humans is essential for identifying more effective strategies for antimicrobial drug treatment and for control of blood glucose levels in diabetic patients with TB. In addition, the influence that IGT and insulin resistance have on TB susceptibility and pathogenesis has not been adequately investigated.Currently, the animal models most often used to study the pathogenesis of TB concurrent with diabetes are inbred strains of mice treated with high doses of the cytotoxic drug streptozotocin (STZ).^7,8 These models mimic chronic hyperglycemia resulting from total insulin deficiency, as in human type 1 diabetes mellitus. Although this strategy has provided valuable information on how absolute insulin deficiency and persistent hyperglycemia affect active TB disease, it fails to take into account a variety of other metabolic defects associated with IGT and type 2 diabetes through dietary manipulation. Unlike murine models, lipid metabolism in the guinea pig more closely resembles that of humans, making it ideal for studying cardiovascular disease risks and other consequences of altered glucose and lipid metabolism associated with type 2 diabetes.⁹ Moreover, most mouse strains used to model TB, including those previously used in diabetes-TB comorbidity studies, fail to respond to M. tuberculosis infection with the development of pulmonary and extrapulmonary granulomata with caseous necrosis, as is typical in humans, as well as guinea pigs, nonhuman primates, and rabbits.¹⁰ The value of the guinea pig TB model has been further validated recently in studies of natural transmission of M. tuberculosis from human patients with TB to guinea pigs. A subpopulation of guinea pigs exposed to aerosols from patients with TB developed active TB disease and an array of clinical and pathologic responses, which more accurately reflects the clinical variation of naturally occurring M. tuberculosis infection in humans.¹¹Clinical studies have shown that the increased susceptibility of diabetic patients to M. tuberculosis is accompanied by an altered host response to infection. Diabetic patients with active TB have higher bacterial burdens based on sputum culture and are refractory to first-line antimicrobial combination therapy, with longer time to sputum conversion and higher mortality rates during therapy.^12–14 Diabetic patients often have atypical radiographic findings, with more frequent involvement of lower lung lobes, and some studies indicate a higher rate of cavitary disease, consistent with more severe pulmonary pathology.^15,16 However, the impact that diabetes has on the development of extrapulmonary TB in humans is conflicting with some studies, indicating a relative risk similar to or less than that of nondiabetic patients, whereas others show a predisposition for extrapulmonary and even miliary disease patterns in diabetic patients.^5,17,18 In patients with TB and poorly controlled type 2 diabetes, an exaggerated innate and type 1 cytokine response has been demonstrated clinically.¹⁹ However, the impact that insulin resistance alone has on the response to M. tuberculosis infection is unknown. The goal of this study was to develop an animal model that more closely mimics the clinical and immunologic manifestations of diabetes-TB comorbidity in humans.     

Association between Mycobacterium tuberculosis Beijing/W lineage strain infection and extrathoracic tuberculosis: Insights from epidemiologic and clinical characterization of the three principal genetic groups of M. tuberculosis clinical isolates

Clinical strains of Mycobacterium tuberculosis can be divided into three principal genetic groups based on the single-nucleotide polymorphisms at the katG gene codon 463 and the gyrA gene codon 95. One subgroup of genetic group 1, the Beijing/W lineage, has been widely studied because of its worldwide distribution and association with outbreaks. In order to increase our understanding of the clinical and epidemiological relevance of the genetic grouping of M. tuberculosis clinical strains and the Beijing/W lineage, we investigated the genetic grouping of 679 clinical isolates of M. tuberculosis, representing 96.3% of culture-confirmed tuberculosis cases diagnosed in Arkansas between January 1996 and December 2000 using PCR and DNA sequencing. We assessed the associations of infections by different genetic groups of M. tuberculosis strains and infection by the Beijing/W lineage strains with the clinical and epidemiological characteristics of the patients using chi-square tests and multivariate logistic regression analysis. Of the 679 study isolates, 676 fell into one of the three principal genetic groups, with 63 (9.3%) in group 1, 438 (64.8%) in group 2, and 175 (25.9%) in group 3. After adjusting for potential confounding of age, gender, race/ethnicity, human immunodeficiency virus serostatus, and plcD genotype in a multivariate logistic regression model, patients infected by the Beijing/W lineage isolates were nearly three times as likely as patients infected with the non-Beijing/W lineage isolates to have an extrathoracic involvement (odds ratio [95% confidence interval], 2.85 [1.33, 6.12]). Thus, the Beijing/W lineage strains may have some special biological features that facilitate the development of extrathoracic tuberculosis.     

Génétique humaine de la tuberculose

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major public health problem worldwide, resulting in 8.7 million new cases and 1.4 million deaths each year. One third of the world's population is exposed to M. tuberculosis and, after exposure, most, but not all, individuals become infected. Among infected subjects, only a minority (∼10%) will eventually develop clinical disease, which is typically either a primary, often extra-pulmonary, TB in children, or a reactivation, pulmonary TB in adults. Considerable genetic epidemiological evidence has accumulated to support a major role for human genetic factors in the development of TB. Numerous association studies with various candidate genes have been conducted in pulmonary TB, with very few consistent results. Recent genome-wide association studies revealed only a modest role for two inter-genic polymorphisms. However, a first major locus for pulmonary TB was mapped to chromosome 8q12-q13 in a Moroccan population after a genome-wide linkage screen. Using a similar strategy, two other major loci controlling TB infection were recently identified. While the precise identification of these major genes is ongoing, the other fascinating observation of these last years was the demonstration that TB can also reflect a Mendelian predisposition. Following the findings obtained in the syndrome of Mendelian susceptibility to mycobacterial diseases, several children with complete IL-12Rβ1 deficiency, were found to have severe TB as their sole phenotype. Overall, these recent findings provide the proof of concept that the human genetics of TB involves a continuous spectrum from Mendelian to complex predisposition with intermediate major gene involvement. The understanding of the molecular genetic basis of TB will have fundamental immunological and medical implications, in particular for the development of new vaccines and treatments.     

Variation in Gamma Interferon Responses to Different Infecting Strains of Mycobacterium tuberculosis in Acid-Fast Bacillus Smear-Positive Patients and Household Contacts in Antananarivo,Madagascar

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop tuberculosis (TB), though many may become latently infected. More precise measurement of the human immune response to M. tuberculosis infection may help us understand this difference and potentially identify those subjects most at risk of developing active disease. Gamma interferon (IFN-γ) production has been widely used as a proxy marker to study infection and to examine the human immune response to specific M. tuberculosis antigens. It has been suggested that genetically distinct M. tuberculosis strains may invoke different immune responses, although how these differences influence the immune responses and clinical outcome in human tuberculosis is still poorly understood. We therefore evaluated the antigen-specific IFN-γ production responses in peripheral blood mononuclear cells from two cohorts of subjects recruited in Antananarivo, Madagascar, from 2004 to 2006 and examined the influence of the infecting M. tuberculosis strains on this response. The cohorts were sputum-positive index cases and their household contacts. Clinical strains isolated from the TB patients were typed by spoligotyping. Comparison of the IFN-γ responses with the spoligotype of the infecting clinical strains showed that “modern” M. tuberculosis strains, like Beijing and Central Asian (CAS) strains, tended to induce lower IFN-γ responses than “ancient” strains, like East African-Indian (EAI) strains, in index cases and their household contacts. These results suggest that new strains may have evolved to induce a host response different from that of ancient strains. These findings could have important implications in the development of therapeutic and diagnostic strategies.Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major cause of global morbidity and mortality throughout the world. It is estimated that there are in excess of new 8 million cases of TB each year, and this represents just the tip of the iceberg. Infection with M. tuberculosis leads to clinically active TB in about 5 to 10% of exposed individuals. A much higher proportion of exposed individuals apparently become latently infected, and these individuals may remain noninfectious and symptom free for years. Approximately one-third of the world population is thought to be latently infected with M. tuberculosis. However, under some circumstances (in about 5% of the latently infected people), the host immune response is perturbed and latent M. tuberculosis infection may develop into clinically active TB (52). This process is most prominent in individuals coinfected with human immunodeficiency virus (HIV), but it can also occur with impairment of the immune system associated with old age, malnutrition, anti-inflammatory drug treatment, etc. Reactivation of latent disease is thought to contribute roughly half of all TB cases, and thus, understanding the factors controlling the development of acute primary TB or latent infection is crucial to TB control (64).Gamma interferon (IFN-γ) production has been widely used to study infection and to examine the human immune response to specific M. tuberculosis antigens. The 6-kDa early secreted antigenic target (ESAT-6) antigen, encoded by genes located within region of difference 1 (RD1) of the M. tuberculosis genome, is much more specific for M. tuberculosis than purified protein derivative (PPD), as these genes were deleted from M. bovis in the development of BCG substrains or are not found in most environmental mycobacteria (29, 53). Some studies showed that the level of IFN-γ release in response to ESAT-6 could identify TB contacts at risk of developing active disease after recent infection (3, 18, 30). CFP7 or TB10.4 is an immunodominant antigen recognized by TB patients and M. bovis BCG-vaccinated subjects, while ESAT-6 is specific to TB patients and induces a strong IFN-γ response (51). Moreover, since CFP7 induces strong protection against infection by M. tuberculosis, it was proposed to be a TB vaccine candidate (1, 19).There is a growing number of observations indicating that TB cases resulting from infection with epidemic strains, such as the W-Beijing strains (22, 35, 39, 44), may display a more severe pathology or more severe symptoms. Beijing strains were also found to induce higher fevers in pulmonary TB patients than other strains (62). In addition, the Beijing genotype, which is responsible for more than 80% of TB cases in China, was associated with virulence and high transmissibility (7, 28). The same has been found more recently with the RD(Rio) strains belonging to the Latin America-Mediterranean (LAM) family (38). Despite the fact that other epidemiological and clinical studies have failed to confirm any association between the mycobacterial genotype and the clinical presentation (8, 41, 43), the immunological aspects of infection with these strains is still of interest and poorly described.Epidemiological studies carried out in Madagascar showed no association between IS6110 patterns and clinical tuberculosis presentation (47), but they did reveal a heterologous population of M. tuberculosis strains, including the existence of a high frequency of unusual genotypes, such as the shared type 109 from the EAI8-MDG family (SpolDB4) (10) or strains with a single copy of IS6110 (24, 46). Since there are limited data on the correlation of the strain genotype with clinical features or the host immune response in patients and their contacts (57, 59), we investigated the IFN-γ response to the ESAT-6, CFP7, and PPD antigens in pulmonary TB patients and their household contacts (as this is commonly used as a biomarker to identify M. tuberculosis infection) and examined the influence of the M. tuberculosis genotype on this response.     

Gamma Interferon Assays Used in the Diagnosis of Tuberculosis

Tuberculosis (TB) is an ancient disease that has infected humans for thousands of years. However, despite diagnostic tests that detect the disease and effective therapy, there are still millions of people worldwide who are infected with TB. The first TB test used to detect infected patients was a skin test that identifies individuals actively infected with TB. This test used a mix of proteins from culture filtrates of the bacteria Mycobacterium tuberculosis. Recently, two new diagnostic tests have been introduced; these two new tests can detect TB infection in patients by challenging peripheral blood cells with specific TB proteins. These assays measure the cellular immune response to these proteins. This minireview evaluates the new assays and compares them to the use of the TB skin test. The use of these new assays may have some advantages in detecting individuals with active tuberculosis. However, there is still a role for the use of the skin test, especially in young patients.     

The re-emergence of tuberculosis: what have we learnt from molecular epidemiology?

Tuberculosis (TB) has re-emerged over the past two decades: in industrialized countries in association with immigration, and in Africa owing to the human immunodeficiency virus epidemic. Drug-resistant TB is a major threat worldwide. The variable and uncertain impact of TB control necessitates not only better tools (diagnostics, drugs, and vaccines), but also better insights into the natural history and epidemiology of TB. Molecular epidemiological studies over the last two decades have contributed to such insights by answering longstanding questions, such as the proportion of cases attributable to recent transmission, risk factors for recent transmission, the occurrence of multiple Mycobacterium tuberculosis infection, and the proportion of recurrent TB cases attributable to re-infection. M. tuberculosis lineages have been identified and shown to be associated with geographical origin. The Beijing genotype is strongly associated with multidrug resistance, and may have escaped from bacille Calmette–Guérin-induced immunity. DNA fingerprinting has quantified the importance of institutional transmission and laboratory cross-contamination, and has helped to focus contact investigations. Questions to be answered in the near future with whole genome sequencing include identification of chains of transmission within clusters of patients, more precise quantification of mixed infection, and transmission probabilities and rates of progression from infection to disease of various M. tuberculosis lineages, as well as possible variations in vaccine efficacy by lineage. Perhaps most importantly, dynamics in the population structure of M. tuberculosis in response to control measures in high-prevalence areas should be better understood.     

Mycobacterium bovis BCG immunization induces protective immunity against nine different Mycobacterium tuberculosis strains in mice

Recent preclinical and epidemiologic studies have suggested that certain Mycobacterium tuberculosis genotypes (in particular, Beijing lineage strains) may be resistant to Mycobacterium bovis BCG vaccine-induced antituberculosis protective immunity. To investigate the strain specificity of BCG-induced protective responses in a murine model of pulmonary tuberculosis, C57BL/6 mice were vaccinated with BCG vaccine and then challenged 2 months later with one of nine M. tuberculosis isolates. Four of these strains were from the W-Beijing lineage (HN878, N4, NHN5, and ChS) while four were non-Beijing-type isolates (C913, CDC1551, NY669, and NY920). As a control, the WHO standard M. tuberculosis Erdman strain was evaluated in these vaccination/challenge experiments. To assess the protective responses evoked by BCG immunization, organ bacterial burdens and lung pathology were assessed in vaccinated and naïve mice at 4, 12, and 20 weeks postchallenge as well as during the day of infection. At 4 weeks after the aerosol challenge with each of these strains, significantly reduced bacterial growth in the lungs and spleens and significantly improved lung pathology were seen in all vaccinated animals compared to naïve controls. After 12 weeks, reduced organ bacterial burdens were detected in vaccinated animals infected with six of nine challenge strains. Although lung CFU values were lower in vaccinated mice for only three of nine groups at 20 weeks postchallenge, significantly decreased lung inflammation was seen in all immunized animals relative to controls at 20 weeks postchallenge. Taken together, these data demonstrate that BCG vaccination protects against infection with diverse M. tuberculosis strains in the mouse model of pulmonary tuberculosis and suggest that strain-specific resistance to BCG-induced protective immunity may be uncommon.     

Assessment of three commercially available serologic assays for detection of antibodies to Mycobacterium tuberculosis and identification of active tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major world disease, with approximately 9 million new cases each year. Identification and treatment of active disease are essential for TB control. Serology may offer increased detection of active disease in patients with a positive tuberculin skin test (TST) or QuantiFERON-TB (QFT-G). The InBios Active TbDetect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), IBL M. tuberculosis IgG ELISA, and Anda Biologics TB ELISAs were evaluated for the ability to detect M. tuberculosis antibodies in patients with active disease. Agreement, sensitivity, and specificity for each ELISA were determined and compared to those for culture or amplified direct detection and M. tuberculosis low-risk control patients. The InBios Active TbDetect ELISA had an agreement of 96.2%, a sensitivity of 83.3%, and a specificity of 98.9%. The IBL M. tuberculosis ELISA had an agreement of 84.0%, a sensitivity of 5.6%, and a specificity of 100.0%. The agreement, sensitivity, and specificity of the Anda Biologics TB ELISA were 74.2%, 83.3%, and 72.0%, respectively. The sensitivity for detecting M. tuberculosis antibodies in human immunodeficiency virus-associated TB was 50% for both the InBios Active TbDetect ELISA and the Anda Biologics TB ELISA and 0% for the IBL M. tuberculosis ELISA. The positivity rates for InBios Active TbDetect ELISA, IBL M. tuberculosis ELISA, and Anda Biologics TB ELISA in latently infected individuals positive by TST and/or QFT-G were 5.1%, 0.0%, and 30.8%, respectively. It can be concluded that the InBios Active TbDetect IgG ELISA is superior to the other ELISAs in accurately detecting active TB.     

Early Changes by 18Fluorodeoxyglucose Positron Emission Tomography Coregistered with Computed Tomography Predict Outcome after Mycobacterium tuberculosis Infection in Cynomolgus Macaques

Cynomolgus macaques infected with low-dose Mycobacterium tuberculosis develop both active tuberculosis and latent infection similar to those of humans, providing an opportunity to study the clinically silent early events in infection. ¹⁸Fluorodeoxyglucose radiotracer with positron emission tomography coregistered with computed tomography (FDG PET/CT) provides a noninvasive method to measure disease progression. We sought to determine temporal patterns of granuloma evolution that distinguished active-disease and latent outcomes. Macaques (n = 10) were infected with low-dose M. tuberculosis with FDG PET/CT performed during infection. At 24 weeks postinfection, animals were classified as having active disease (n = 3) or latent infection (n = 6), with one “percolator” monkey. Imaging characteristics (e.g., lesion number, metabolic activity, size, mineralization, and distribution of lesions) were compared among active and latent groups. As early as 3 weeks postinfection, more pulmonary granulomas were observed in animals that would later develop active disease than in those that would develop latent infection. Over time, new lesions developed in active-disease animals but not in latent animals. Granulomas and mediastinal lymph nodes from active-disease but not latent animals consistently increased in metabolic activity at early time points. The presence of fewer lesions at 3 weeks and the lack of new lesion development in animals with latent infection suggest that innate and rapid adaptive responses are critical to preventing active tuberculosis. A greater emphasis on innate responses and/or rapid recruitment of adaptive responses, especially in the airway, should be emphasized in newer vaccine strategies.     

Protein kinase C-delta (PKCδ), a marker of inflammation and tuberculosis disease progression in humans,is important for optimal macrophage killing effector functions and survival in mice

We previously demonstrated that protein kinase C-δ (PKCδ) is critical for immunity against Listeria monocytogenes, Leishmania major, and Candida albicans infection in mice. However, the functional relevance of PKCδ during Mycobacterium tuberculosis (Mtb) infection is unknown. PKCδ was significantly upregulated in whole blood of patients with active tuberculosis (TB) disease. Lung proteomics further revealed that PKCδ was highly abundant in the necrotic and cavitory regions of TB granulomas in multidrug-resistant human participants. In murine Mtb infection studies, PKCδ^−/− mice were highly susceptible to tuberculosis with increased mortality, weight loss, exacerbated lung pathology, uncontrolled proinflammatory cytokine responses, and increased mycobacterial burdens. Moreover, these mice displayed a significant reduction in alveolar macrophages, dendritic cells, and decreased accumulation of lipid bodies (lungs and macrophages) and serum fatty acids. Furthermore, a peptide inhibitor of PKCδ in wild-type mice mirrored lung inflammation identical to infected PKCδ^−/− mice. Mechanistically, increased bacterial growth in macrophages from PKCδ^−/− mice was associated with a decline in killing effector functions independent of phagosome maturation and autophagy. Taken together, these data suggest that PKCδ is a marker of inflammation during active TB disease in humans and required for optimal macrophage killing effector functions and host protection during Mtb infection in mice.     

MicroRNA-31 mediated by interferon regulatory factor 7 signaling facilitates control of Mycobacterium tuberculosis infection

Tuberculosis (TB) induced by Mycobacterium tuberculosis (M. tuberculosis) infection remains a global most deadly infectious disease. While development of more effective TB vaccines and therapeutics relies on identifications of true biomarkers designating an immune protection against M. tuberculosis infection, exact protective immune components against M. tuberculosis infection remain largely unidentified. We previously found that severe TB induced remarkable up-regulation of interferon regulatory factor 7 (IRF7) and IRF7-related gene signatures, implicating that some unknown downstream molecules in IRF7 signaling cascades may determine the M. tuberculosis infection outcomes and serve as a protective immune component against M. tuberculosis infection. Indeed, here, we observe that genetic ablation of IRF7 leads to more severe lung pathology, increased M. tuberculosis burdens, impaired differentiation of effector/memory T subsets, and extensively elevated expression of pro-inflammatory cytokines in lungs. Importantly, IRF7 is vital for sustaining expression of PD-1/PD-L1 and PD-1/PD-L1-modulated miRNA-31. Moreover, interventions of miRNA-31 expressions via administration of miRNA-31 agomir reduces lung pathology and bacilli burdens via inducing up-regulation of gene sets involved in biological processes of defense response or cellular and chemical homeostasis in lungs. Thus, this study uncovers previously unrecognized importance and mechanisms of IRF7-mediated miRNA-31 as a protective immune component against M. tuberculosis infection.     

Infection of great apes and a zoo keeper with the same Mycobacterium tuberculosis spoligotype

An animal keeper was diagnosed with pulmonary tuberculosis (TB) after bi-annual screening for latent TB infection in zoo employees. In the same period, several bonobos of the zoo were suffering from TB as well. The Mycobacterium tuberculosis strains from both the animal keeper and the bonobos appeared identical. We provide evidence that the animals infected their keeper.     

Progression of chronic pulmonary tuberculosis in mice intravenously infected with ethambutol resistant Mycobacterium tuberculosis

Purpose: Ethambutol (EMB) is an important first line drug, however little information on its molecular mechanism of resistance and pathogenicity of resistant isolates is available. Present work was designed to study virulence of the EMB resistant M. tuberculosis strains and the host responses in-vivo on infection of EMB resistant M. tuberculosis using Balb/c mouse model of infection. Methods: Three groups of Balb/c mice (female, age 4-6 wk; 21 mice in each group) were infected intravenously with 106 CFU of M. tuberculosis H37Rv and two EMB resistant clinical isolates. Age and sex matched control animals were mock inoculated with Middlebrook 7H9 broth alone. At 10, 20, 30, 40, 50, 60, and 70 days post-infection three animals from each group were sacrificed by cervical dislocation and lung tissue was collected for further analysis. Results: Infection with EMB resistant M. tuberculosis led to progressive and chronic disease with significantly high bacillary load (p=0.02). Massive infiltration and exacerbated lung pathology with increased expression of IFN-γand TNF-αwas observed in lungs of mice infected with EMB resistant strains. The present study suggests that infection with EMB resistant M. tuberculosis leads to chronic infection with subsequent loss of lung function, bacterial persistence with elevated expression of TNF-αresulting in increased lung pathology. Conclusion: These findings highlight that EMB resistant M. tuberculosis regulates host immune response differentially and its pathogenicity is different from drug sensitive strains of M. tuberculosis.     

Immune Responses in Cattle Inoculated with Mycobacterium bovis,Mycobacterium tuberculosis,or Mycobacterium kansasii

Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare the antigen-specific immune responses to various patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis infection) and colonization without pathology (M. tuberculosis infection) to no colonization or pathology (M. kansasii infection). Delayed-type hypersensitivity and gamma interferon responses were elicited by each mycobacterial inoculation; however, the responses by the M. bovis- and M. tuberculosis-inoculated animals exceeded those of the M. kansasii-inoculated animals. Specific antibody responses were detected in all M. tuberculosis- and M. bovis-inoculated cattle 3 weeks after inoculation. From 6 to 16 weeks after M. tuberculosis inoculation, the antibody responses waned, whereas the responses persisted with M. bovis infection. With M. kansasii inoculation, initial early antibody responses waned by 10 weeks after inoculation and then increased 2 weeks after the injection of purified protein derivative for the skin test at 18 weeks after challenge. These findings indicate that antibody responses are associated with the antigen burden rather than the pathology, cellular immune responses to tuberculin correlate with infection but not necessarily with the pathology or bacterial burden, and exposure to mycobacterial antigens may elicit an antibody response in a presensitized animal.Tuberculosis (TB) in humans and animals may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedi, M. microti, M. caprae, or M. canetti [8]). Despite their ∼99.95% sequence identity (12), M. bovis and M. tuberculosis exhibit distinct differences in virulence and host adaptation. Compared to M. tuberculosis, experimental M. bovis infection of mice or rabbits results in a more severe pathology and shorter mean survival times (9, 17, 18). Mycobacterium tuberculosis is primarily a human pathogen that demonstrates a high level of attenuation in cattle (as reviewed by Francis in 1947 [10]), whereas M. bovis has a wider host range and affects many domesticated and free-ranging mammals as well as humans. Prior to the initiation of control and eradication campaigns in the early to mid-1900s, M. bovis infection accounted for up to 30% of human tuberculosis cases, with M. bovis being transmitted to humans primarily by the consumption of unpasteurized dairy products and contact with infected livestock. Control efforts, including slaughter surveillance and test/cull campaigns, have dramatically reduced the prevalence of M. bovis infection in domestic cattle herds, thereby reducing the spread of M. bovis to humans. However, in developing countries, M. bovis infection of humans persists as a serious and relatively common zoonosis (16).Although Mycobacterium kansasii is not a member of the M. tuberculosis complex, it may cause disease in otherwise healthy humans, albeit infrequently, that is clinically indistinguishable from M. tuberculosis infection (1, 3). As with humans, M. kansasii infection of cattle is uncommon; however, it is occasionally associated with granulomatous lesions within lymph nodes and the respiratory tract of cattle (B. Harris, unpublished observations). Of particular relevance for the diagnosis of tuberculosis, M. kansasii infection/sensitization may elicit responses to antigens generally considered to be tuberculosis specific, such as ESAT-6, CFP-10, and MPB83 (2, 30, 35).With experimental M. bovis infection of cattle, the levels of MPB83-specific antibody correlate with disease severity, bacterial burden, and specific cell-mediated immune responses (15, 33). With this particular scenario, disease severity (i.e., pathology) and bacterial burden are intimately linked; thus, it is difficult to define a potential correlation of a particular immune response to either readout independently. Prior studies have demonstrated that virulent and attenuated strains of M. bovis induce similar delayed-type hypersensitivity responses in cattle; however, only the virulent M. bovis strain induces a persistent gamma interferon (IFN-γ), interleukin-2 (IL-2), and antibody response (34). The objective of the present study was to compare mycobacterium-specific immune responses to the patterns of mycobacterial disease expression in which the mycobacterial burden is uncoupled from pathological changes. Disease expression patterns included persistent colonization with an associated pathology (i.e., M. bovis infection), colonization without an associated pathology (i.e., M. tuberculosis infection), and no colonization or pathology (i.e., M. kansasii infection). Antigen-specific immune responses were evaluated for their correlation to manifestations of disease expression.     

Effects of host-directed therapies on the pathology of tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has co-evolved with the human immune system and utilizes multiple strategies to persist within infected cells, to hijack several immune mechanisms, and to cause severe pathology and tissue damage in the host. This delays the efficacy of current antibiotic therapy and contributes to the evolution of multi-drug-resistant strains. These challenges led to the development of the novel approach in TB treatment that involves therapeutic targeting of host immune response to control disease pathogenesis and pathogen growth, namely, host-directed therapies (HDTs). Such HDT approaches can (1) enhance the effect of antibiotics, (2) shorten treatment duration for any clinical form of TB, (3) promote development of immunological memory that could protect against relapse, and (4) ameliorate the immunopathology including matrix destruction and fibrosis associated with TB. In this review we discuss TB-HDT candidates shown to be of clinical relevance that thus could be developed to reduce pathology, tissue damage, and subsequent impairment of pulmonary function. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Profiling antibodies to Mycobacterium tuberculosis by multiplex microbead suspension arrays for serodiagnosis of tuberculosis

Tuberculosis (TB) is a serious global disease. The fatality rate attributed to TB is among the highest of infectious diseases, with approximately 2 million deaths occurring per year worldwide. Identification of individuals infected with Mycobacterium tuberculosis and screening of their immediate contacts is crucial for controlling the spread of TB. Current methods for detection of M. tuberculosis infection are not efficient, in particular, for testing large numbers of samples. We report a novel and efficient multiplex microbead immunoassay (MMIA), based on Luminex technology, for profiling antibodies to M. tuberculosis. Microbead sets identifiable by unique fluorescence were individually coated with each of several M. tuberculosis antigens and tested in multiplex format for antibody detection in the experimental nonhuman primate model of TB. Certain M. tuberculosis antigens, e.g., ESAT-6, CFP-10, and HspX, were included to enhance the specificity of the MMIA, because these antigens are absent in nontuberculous mycobacteria and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. The MMIA enabled simultaneous detection of multiple M. tuberculosis plasma antibodies in several cohorts of macaques representing different stages of infection and/or disease. Antibody profiles were defined in early and latent/chronic infection. These proof-of-concept findings demonstrate the potential clinical use of the MMIA. In addition, the MMIA serodetection system has a potential for mining M. tuberculosis open reading frames (about 4,000) to discover novel target proteins for the development of more-comprehensive TB serodiagnostic tests.     

Protection and Long-Lived Immunity Induced by the ID93/GLA-SE Vaccine Candidate against a Clinical Mycobacterium tuberculosis Isolate

Mycobacterium tuberculosis HN878 represents a virulent clinical strain from the W-Beijing family, which has been tested in small animal models in order to study its virulence and its induction of host immune responses following infection. This isolate causes death and extensive lung pathology in infected C57BL/6 mice, whereas lab-adapted strains, such as M. tuberculosis H37Rv, do not. The use of this clinically relevant isolate of M. tuberculosis increases the possibilities of assessing the long-lived efficacy of tuberculosis vaccines in a relatively inexpensive small animal model. This model will also allow for the use of knockout mouse strains to critically examine key immunological factors responsible for long-lived, vaccine-induced immunity in addition to vaccine-mediated prevention of pulmonary immunopathology. In this study, we show that the ID93/glucopyranosyl lipid adjuvant (GLA)-stable emulsion (SE) tuberculosis vaccine candidate, currently in human clinical trials, is able to elicit protection against M. tuberculosis HN878 by reducing the bacterial burden in the lung and spleen and by preventing the extensive lung pathology induced by this pathogen in C57BL/6 mice.