Human TB Research Articles

Mycobacteria that cause tuberculosis have retained ancestrally acquired genes for the biosynthesis of chemically diverse terpene nucleosides.

Mycobacterium tuberculosis (Mtb) releases the unusual terpene nucleoside 1-tuberculosinyladenosine (1-TbAd) to block lysosomal function and promote survival in human macrophages. Using conventional approaches, we found that genes Rv3377c and Rv3378c, but not Rv3376, were necessary for 1-TbAd biosynthesis. Here, we introduce linear models for mass spectrometry (limms) software as a next-generation lipidomics tool to study the essential functions of lipid biosynthetic enzymes on a whole-cell basis. Using limms, whole-cell lipid profiles deepened the phenotypic landscape of comparative mass spectrometry experiments and identified a large family of approximately 100 terpene nucleoside metabolites downstream of Rv3378c. We validated the identity of previously unknown adenine-, adenosine-, and lipid-modified tuberculosinol-containing molecules using synthetic chemistry and collisional mass spectrometry, including comprehensive profiling of bacterial lipids that fragment to adenine. We tracked terpene nucleoside genotypes and lipid phenotypes among Mycobacterium tuberculosis complex (MTC) species that did or did not evolve to productively infect either human or nonhuman mammals. Although 1-TbAd biosynthesis genes were thought to be restricted to the MTC, we identified the locus in unexpected species outside the MTC. Sequence analysis of the locus showed nucleotide usage characteristic of plasmids from plant-associated bacteria, clarifying the origin and timing of horizontal gene transfer to a pre-MTC progenitor. The data demonstrated correlation between high level terpene nucleoside biosynthesis and mycobacterial competence for human infection, and 2 mechanisms of 1-TbAd biosynthesis loss. Overall, the selective gain and evolutionary retention of tuberculosinyl metabolites in modern species that cause human TB suggest a role in human TB disease, and the newly discovered molecules represent candidate disease-specific biomarkers.

Lipid Peroxidation and Type I Interferon Coupling Fuels Pathogenic Macrophage Activation Causing Tuberculosis Susceptibility.

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop pulmonary TB. We developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: the development of necrotic lung granulomas and determined that the sst1-susceptible phenotype was driven by the aberrant macrophage activation. This study demonstrates that the aberrant response of the sst1-susceptible macrophages to prolonged stimulation with TNF is primarily driven by conflicting Myc and antioxidant response pathways leading to a coordinated failure 1) to properly sequester intracellular iron and 2) to activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled IFNβ superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. The upregulation of Myc pathway in peripheral blood cells of human TB patients was significantly associated with poor outcomes of TB treatment. Thus, Myc dysregulation in activated macrophages results in an aberrant macrophage activation and represents a novel target for host-directed TB therapies.

Spatial distribution of Mycobacterium tuberculosis mRNA and secreted antigens in acid-fast negative human antemortem and resected tissue

The ability to detect evidence of Mycobacterium tuberculosis (Mtb) infection within human tissues is critical to the study of Mtb physiology, tropism, and spatial distribution within TB lesions. The capacity of the widely-used Ziehl-Neelsen (ZN) staining method for identifying Mtb acid-fast bacilli (AFB) in tissue is highly variable, which can limit detection of Mtb bacilli for research and diagnostic purposes. Here, we sought to circumvent these limitations via detection of Mtb mRNA and secreted antigens in human tuberculous tissue. We adapted RNAscope, an RNA in situ hybridisation (RISH) technique, to detect Mtb mRNA in ante- and postmortem human TB tissues and developed a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). We identified Mtb mRNA within intact and disintegrating bacilli as well as extrabacillary mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchiolar epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. RNAscope and dual ZN/immunohistochemistry staining are well-suited for identifying subsets of intact Mtb and/or bacillary remnants in human tissue. RNAscope can identify Mtb mRNA in ZN-negative tissues from patients with TB and may have diagnostic potential in complex TB cases. Wellcome Leap Delta Tissue Program, Wellcome Strategic Core Award, the National Institutes of Health (NIH, USA), the Mary Heersink Institute for Global Health at UAB, the UAB Heersink School of Medicine.

Human Disease due to Mycobacterium bovis Linked to Free-Ranging Deer in Michigan.

A unique enzootic focus of Mycobacterium bovis in free-ranging deer was identified in northern lower Michigan in 1994, with subsequent evidence of transmission to local cattle herds. Between 2002 and 2017, 3 Michigan deer hunters with M. bovis disease were previously reported. We present 4 additional human cases linked to the zoonotic focus in deer, utilizing genomic epidemiology to confirm close molecular associations among human, deer and cattle M. bovis isolates. Identification of human tuberculosis (TB) cases with cultures of M. bovis was provided from the Michigan Department of Health and Human Services (MDHHS) tuberculosis database. Clinical review and interviews focused on risk factors for contact with wildlife and cattle. Whole genome sequences of human isolates were compared with a veterinary library of M. bovis strains to identify those linked to the enzootic focus. Three confirmed and 1 probable human case with M. bovis disease were identified between 2019 and 2022, including cutaneous disease, 2 severe pulmonary disease cases, and human-to-human transmission. The 3 human isolates had 0-3 single-nucleotide polymorphisms (SNPs) with M. bovis strains circulating in wild deer and domestic cattle in Michigan. Spillover of enzootic M. bovis from deer to humans and cattle continues to occur in Michigan. Future studies should examine the routes of transmission and degree of risk to humans through expanded epidemiological surveys. A One Health approach linking human, veterinary and environmental health should address screening for TB infection, public education, and mitigation of transmission.

Engineering a dual vaccine against COVID-19 and tuberculosis.

The COVID-19 pandemic, caused by SARS-CoV-2 virus, has been one of the top public health threats across the world over the past three years. Mycobacterium bovis BCG is currently the only licensed vaccine for tuberculosis, one of the deadliest infectious diseases in the world, that is caused by Mycobacterium tuberculosis. In the past decades, recombinant M.bovis BCG has been studied as a novel vaccine vector for other infectious diseases in humans besides tuberculosis, such as viral infections. In the current study, we generated a recombinant M. bovis BCG strain AspikeRBD that expresses a fusion protein consisting of M. tb Ag85A protein and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using synthetic biology technique. Our results show that the recombinant M. bovis BCG strain successfully expressed this fusion protein. Interestingly, the recombinant M. bovis BCG strain AspikeRBD significantly induced SARS-CoV-2 spike-specific T cell activation and IgG production in mice when compared to the parental M.bovis BCG strain, and was more potent than the recombinant M.bovis BCG strain expressing SARS-CoV-2 spike RBD alone. As expected, the recombinant M. bovis BCG strain AspikeRBD activated an increased number of M. tb Ag85A-specific IFNγ-releasing T cells and enhanced IgG production in mice when compared to the parental M.bovis BCG strain or the BCG strain expressing SARS-CoV-2 spike RBD alone. Taken together, our results indicate a potential application of the recombinant M. bovis BCG strain AspikeRBD as a novel dual vaccine against SARS-CoV-2 and M. tb in humans.

Assessment of Co-Infection in Tuberculosis Patients Prior to their Enrollment to the Direct Observed Treatment (Dot) Program in Buea, Limbe, and Douala Tuberculosis Treatment Centers

Introduction: Co-infections during Tuberculosis infection are among the leading causes of morbidity and mortality worldwide. These infections are a major public health problem among patients in most developing countries, including Cameroon. One of the important public problems in coendemic areas is co-infection with TB in humans. Moreover, there is no adequate research hitherto that provides information on the level of co-infection in Tuberculosis patients. Therefore, this study aimed to assess the prevalence and intensity of co-infections in newly diagnosed Tuberculosis patients prior to their enrollment to the Direct Observed Treatment (DOT) program in Buea, Limbe and Douala Tuberculosis treatment centers. Methods: This cross-sectional study was conducted from March to November 2020 using a purposive sampling technique. A fresh sputum sample was collected, processed, and examined through a smear microscopy and culturing technique. Blood samples were collected through finger pricking for the detection of blood parasites using thin and thick blood smears. Stool samples were also collected and processed using a direct wet mount, and Kato-Katz and Modified Ziehl Neelsen staining methods. The data was analyzed using SPSS version 20.0. A Chi-square test was done to assess the prevalence of co-infection among all TB patients recruited for this study. Results: A total of 200 newly diagnosed TB-positive patients were recruited from the three study sites. Out of the total participants recruited, 29.0% were diagnosed positive for blood parasites, 2.0% for intestinal parasites and 15.0% for viral infections, bringing the overall prevalence of co-infection to 46.0%. When specific infections were considered, malaria, filaria, intestinal parasites, HIV and HBV constituted 28.0%, 1.0%, 2.0%, 14.5% and 0.5% respectively. When dual-infection was considered, the results were 0.5% malaria/filaria, 1.5% malaria/intestinal parasites and 2.0% malaria/HIV. The mean parasite densities for all samples diagnosed positive for malaria, filarial and intestinal parasites were 107.0 ± 338.8 parasites/uL, 25.7 ± 356.4 mf/mL and 85.0 ± 85.4 EPG, respectively. Conclusion: The results indicate that there is an overlap of co-infections in TB patients with their interactions expected to lead to complications and difficulty when managing the TB patients, calling for more research and recommending more assessment for co-infection in TB patients before starting treatment.

B cells promote granulomatous inflammation during chronic Mycobacterium tuberculosis infection in mice.

The current study reveals that in chronic TB, the B cell-deficient μMT strain, relative to wild-type (WT) C57BL/6 mice, displays in the lungs lower levels of inflammation that are associated with decreased CD4+ T cell proliferation, diminished Th1 response, and enhanced levels of interleukin (IL)-10. The latter result raises the possibility that B cells may restrict lung expression of IL-10 in chronic TB. These observations are recapitulated in WT mice depleted for B cells using anti-CD20 antibodies. IL-10 receptor (IL-10R) blockade reverses the phenotypes of decreased inflammation and attenuated CD4+ T cell responses in B cell-depleted mice. Together, these results suggest that in chronic murine TB, B cells, by virtue of their capacity to restrict expression of the anti-inflammatory and immunosuppressive IL-10 in the lungs, promote the development of a robust protective Th1 response, thereby optimizing anti-TB immunity. This vigorous Th1 immunity and restricted IL-10 expression may, however, allow the development of inflammation to a level that can be detrimental to the host. Indeed, decreased lung inflammation observed in chronically infected B cell-deficient mice, which exhibit augmented lung IL-10 levels, is associated with a survival advantage relative to WT animals. Collectively, the results reveal that in chronic murine TB, B cells play a role in modulating the protective Th1 immunity and the anti-inflammatory IL-10 response, which results in augmentation of lung inflammation that can be host-detrimental. Intriguingly, in tuberculous human lungs, conspicuous B cell aggregates are present in close proximity to tissue-damaging lesions manifesting necrosis and cavitation, suggesting the possibility that in human TB, B cells may contribute to the development of exacerbated pathology that is known to promote transmission. Since transmission is a major hindrance to TB control, investigating into whether B cells can shape the development of severe pulmonic pathological responses in tuberculous individuals is warranted.

Polymorphisms and gene expression of Notch4 in pulmonary tuberculosis.

Tuberculosis (TB) is a serious public health problem to human health, but the pathogenesis of TB remains elusive. To identify novel candidate genes associated with TB susceptibility, we performed a population-based case control study to genotype 41SNPs spanning 21 genes in 435 pulmonary TB patients and 375 health donors from China. We found Notch4 gene rs206018 and rs422951 polymorphisms were associated with susceptibility to pulmonary tuberculosis. The association was validated in another independent cohort including 790 TB patients and 1,190 healthy controls. Moreover, we identified that the rs206018 C allele was associated with higher level of Notch4 in PBMCs from pulmonary TB patients. Furthermore, Notch4 expression increased in TB patients and higher Notch4 expression correlated with the severer pulmonary TB. Finally, we explored the origin and signaling pathways involved in the regulation of Notch4 expression in response to Mycobacterium tuberculosis (Mtb) infection. We determine that Mtb induced Notch4 and its ligand Jagged1expression in macrophages, and Notch4 through TLR2/P38 signaling pathway and Jagged1 through TLR2/ERK signaling pathway. Our work further strengthens that Notch4 underlay an increased risk of TB in humans and is involved in the occurrence and development of TB, which could serve as a novel target for the host-targeted therapy of TB.

Tuberculosis Vaccines for Cattle

Abstract Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that mainly affects cattle. The traditional bTB control strategy is to slaughter animals positive for the official diagnostic test. This method, however, may not be economically feasible in developing countries and is less effective when cattle are in contact with wildlife reservoirs of infection. In these scenarios, cattle vaccination could be an important strategy to control bTB. In this review, we describe the diversified vaccine development against bTB and identify gaps in current knowledge. We discuss progress made in live attenuated (including BCG), inactivated and subunit vaccines tested in preclinical models and field trials. We also provide an overview of existing knowledge in the correlates and biomarkers of protection after vaccination against bTB, as well as the most recent advances in the understanding of the protective immune response against tuberculosis. The similarities between the mechanisms of the immune protection elicited by humans and cattle against TB and bTB, respectively, make any advance in human TB and bTB vaccines mutually beneficial. In accordance with the One Health concept, this mutual benefit is expected to progress in the future.

The Galton–Watson branching process for TB dynamics: The potential for disease persistence or extinction

Tuberculosis is a critical problem particularly in developing countries of Africa that has resulted in a high mortality rate. The disease is transmitted through air when a susceptible individual inhales Mycobacterium tuberculosis bacteria that have been released by an infected person during coughing, sneezing, singing or speaking. In this work, we formulate and analyze a deterministic model and its corresponding continuous time Markov chain (CTMC) stochastic model to study the dynamics of tuberculosis in humans. The Galton–Watson multitype branching process is adopted to compute the stochastic threshold ρ(M) and determine the possibility of tuberculosis persistence or extinction. The next generation method is employed to find the basic reproduction number R0 and the partial rank correlation coefficient method is used to determine the most sensitive parameters in the spread of tuberculosis. Sensitivity analysis shows that the probability of TB infection, the fraction of individuals who progress to pulmonary TB, pulmonary TB induced death and human recruitment rate influence the transmission of TB disease. If the basic reproduction number R0<1 and the stochastic threshold ρ(M)<1 then tuberculosis clears in human population. If R0>1 then tuberculosis persists in the population. However, there is a possibility of tuberculosis to persist or clear if ρ(M)>1 depending on the number of infectious agents at the beginning of the tuberculosis outbreak. Results reveal that the probability of tuberculosis extinction is small if TB emerges from individuals with pulmonary TB and it is large if TB emerges from individuals with latent TB. If TB emerges from individuals with extra-pulmonary TB, then the disease clears in the population. These results suggest that it is essential to treat humans with pulmonary tuberculosis particularly at the beginning of the disease outbreak so as to control human TB.

System-wide identification of myeloid markers of TB disease and HIV-induced reactivation in the macaque model of Mtb infection and Mtb/SIV co-infection.

Mycobacterium tuberculosis (Mtb) has developed specialized mechanisms to parasitize its host cell, the macrophage. These mechanisms allow it to overcome killing by oxidative burst and persist in the wake of an inflammatory response. Mtb infection in the majority of those exposed is controlled in an asymptomatic form referred to as latent tuberculosis infection (LTBI). HIV is a well-known catalyst of reactivation of LTBI to active TB infection (ATB). Through the use of nonhuman primates (NHPs) co-infected with Mtb and Simian Immunodeficiency Virus (Mtb/SIV), we are able to simulate human progression of TB/AIDS comorbidity. The advantage of NHP models is that they recapitulate the breadth of human TB outcomes, including immune control of infection, and loss of this control due to SIV co-infection. Identifying correlates of immune control of infection is important for both vaccine and therapeutics development. Using macaques infected with Mtb or Mtb/SIV and with different clinical outcomes we attempted to identify signatures between those that progress to active infection after SIV challenge (reactivators) and those that control the infection (non-reactivators). We particularly focused on pathways relevant to myeloid origin cells such as macrophages, as these innate immunocytes have an important contribution to the initial control or the lack thereof, following Mtb infection. Using bacterial burden, C-reactive protein (CRP), and other clinical indicators of disease severity as a guide, we were able to establish gene signatures of host disease state and progression. In addition to gene signatures, clustering algorithms were used to differentiate between host disease states and identify relationships between genes. This allowed us to identify clusters of genes which exhibited differential expression profiles between the three groups of macaques: ATB, LTBI and Mtb/SIV. The gene signatures were associated with pathways relevant to apoptosis, ATP production, phagocytosis, cell migration, and Type I interferon (IFN), which are related to macrophage function. Our results suggest novel macrophage functions that may play roles in the control of Mtb infection with and without co-infection with SIV. These results particularly point towards an interplay between Type I IFN signaling and IFN-γ signaling, and the resulting impact on lung macrophages as an important determinant of progression to TB.

Material and nanomechanical properties of bone structural units of cortical and trabecular iliac bone tissues from untreated postmenopausal osteoporotic women

The differences in bone nanomechanical properties between cortical (Ct) and trabecular (Tb) bone remain uncertain, whereas knowing the respective contribution of each compartment is critical to understand the origin of bone strength. Our purpose was to compare bone mechanical and intrinsic properties of Ct and Tb compartments, at the bone structural unit (BSU) level, in iliac bone taken from a homogeneous untreated human population.Among 60 PMMA-embedded transiliac bone biopsies from untreated postmenopausal osteoporotic women (64 ± 7 year-old), >2000 BSUs were analysed by nanoindentation in physiological wet conditions [indentation modulus (elasticity), hardness, dissipated energy], by Fourier transform infrared (FTIRM) and Raman microspectroscopy (mineral and organic characteristics), and by X-ray microradiography (degree of mineralization of bone, DMB). BSUs were categorized based on tissue age, osteonal (Ost) and interstitial (Int) tissues location and bone compartments (Ct and Tb).Indentation modulus was higher in Ct than in Tb BSUs, both in Ost and Int. dissipated energy was higher in Ct than Tb, in Int BSUs. Hardness was not different between Ct and Tb BSUs. In Ost or Int BSUs, mineral maturity (conversion of non-apatitic into apatitic phosphates) was higher in Ct than in Tb, as well as for collagen maturity (Ost). Mineral content assessed as mineral/matrix (FTIRM and Raman) or as DMB, was lower in Ct than in Tb. Crystallinity (FTIRM) was similar in BSUs from Ct and Tb, and slightly lower in Ct than in Tb when measured by Raman, indicating that the crystal size/perfection was quite similar between Ct and Tb BSUs. The differences found between Ost and Int tissues were much higher than the difference found between Ct and Tb for all those bone material properties. Multiple regression analysis showed that Indentation modulus and dissipated energy were mainly explained by mineral maturity in Ct and by collagen maturity in Tb, and hardness by mineral content in both Ct and Tb.In conclusion, in untreated human iliac bone, Ct and Tb BSUs exhibit different characteristics. Ct BSUs have higher indentation modulus, dissipated energy (Int), mineral and organic maturities than Tb BSUs, without difference in hardness. Although those differences are relatively small compared to those found between Ost and Int BSUs, they may influence bone strength at macroscale.

Identification of region of difference and H37Rv-related deletion in Mycobacterium tuberculosis complex by structural variant detection and genome assembly.

Mycobacterium tuberculosis complex (MTBC), the main cause of TB in humans and animals, is an extreme example of genetic homogeneity, whereas it is still nevertheless separated into various lineages by numerous typing methods, which differ in phenotype, virulence, geographic distribution, and host preference. The large sequence polymorphism (LSP), incorporating region of difference (RD) and H37Rv-related deletion (RvD), is considered to be a powerful means of constructing phylogenetic relationships within MTBC. Although there have been many studies on LSP already, focusing on the distribution of RDs in MTBC and their impact on MTB phenotypes, a crumb of new lineages or sub-lineages have been excluded and RvDs have received less attention. We, therefore, sampled a dataset of 1,495 strains, containing 113 lineages from the laboratory collection, to screen for RDs and RvDs by structural variant detection and genome assembly, and examined the distribution of RvDs in MTBC, including RvD2, RvD5, and cobF region. Consistent with genealogical delineation by single nucleotide polymorphism (SNP), we identified 125 RDs and 5 RvDs at the species, lineage, or sub-lineage levels. The specificities of RDs and RvDs were further investigated in the remaining 10,218 strains, suggesting that most of them were highly specific to distinct phylogenetic groups, could be used as stable genetic markers in genotyping. More importantly, we identified 34 new lineage or evolutionary branch specific RDs and 2 RvDs, also demonstrated the distribution of known RDs and RvDs in MTBC. This study provides novel details about deletion events that have occurred in distinct phylogenetic groups and may help to understand the genealogical differentiation.

A Short History of the BCG Vaccine

BCG vaccine continues to be controversial, live attenuated BCG is still the only vaccine in use which is able to prevent TB in humans. It is still difficult to determine which strain should be used and further detailed analysis of the genomics and immunogenicity of BCG sub-strains may provide an answer to this important question. The only vaccine available is the BCG (Bacille Calmette-Guerin), that has been used for about 100 years, with remarkable results. The majority of the world followed the lead of Europe and the WHO and introduced routine BCG vaccination according to various schedules. According to the Methodological Guide for the Implementation of the National Program for the Prevention, Surveillance and Control of Tuberculosis, BCG vaccination is indicated for all newborns, at the age of 2-7 days (if there are no contraindications), before discharge from maternity and without preliminary tuberculin test.

Bovine tuberculosis in Taiwan, 2008-2019.

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. The impact of bTB on global TB control has been underestimated. We adopted the One Health approach to human bTB surveillance in Taiwan. Of 20,972 human TB cases, 202 (1.0%) were bTB, 78.2% were in males, 85.1% were new cases, 83.2% were pulmonary TB, and most were in Central (52.5%) and Southern (24.8%) Taiwan. Only 18.8% of bTB patients had known animal contact. Of the 202 human M. bovis strains, 100% were resistant to pyrazinamide (PZA), 30.2% were concurrently resistant to isoniazid (INH) and 2.0% were multidrug resistant, defined as being resistant to at least INH and rifampin. Whereas, of the 22 animal M. bovis strains, 100% and 22.7% were resistant to PZA and INH, respectively. Seven spoligotypes and 25 mycobacterial interspersed repetitive unit genotypes were identified. The predominant genotype, SB0265, was also prevalent in livestock. Notably, six animal-specific M. bovis genotypes were identified. bTB differential diagnosis and drug resistance detection are crucial for TB control. Comprehensive surveillance and human-animal interface investigations are needed.

Mycobacterium tuberculosis Specific Protein Rv1509 Evokes Efficient Innate and Adaptive Immune Response Indicative of Protective Th1 Immune Signature.

Dissecting the function(s) of proteins present exclusively in Mycobacterium tuberculosis (M.tb) will provide important clues regarding the role of these proteins in mycobacterial pathogenesis. Using extensive computational approaches, we shortlisted ORFs/proteins unique to M.tb among 13 different species of mycobacteria and identified a hypothetical protein Rv1509 as a ‘signature protein’ of M.tb. This unique protein was found to be present only in M.tb and absent in all other mycobacterial species, including BCG. In silico analysis identified numerous putative T cell and B cell epitopes in Rv1509. Initial in vitro experiments using innate immune cells demonstrated Rv1509 to be immunogenic with potential to modulate innate immune responses. Macrophages treated with Rv1509 exhibited higher activation status along with substantial release of pro-inflammatory cytokines. Besides, Rv1509 protein boosts dendritic cell maturation by increasing the expression of activation markers such as CD80, HLA-DR and decreasing DC-SIGN expression and this interaction was mediated by innate immune receptor TLR2. Further, in vivo experiments in mice demonstrated that Rv1509 protein promotes the expansion of multifunctional CD4+ and CD8+T cells and induces effector memory response along with evoking a canonical Th1 type of immune response. Rv1509 also induces substantial B cell response as revealed by increased IgG reactivity in sera of immunized animals. This allowed us to demonstrate the diagnostic efficacy of this protein in sera of human TB patients compared to the healthy controls. Taken together, our results reveal that Rv1509 signature protein has immunomodulatory functions evoking immunological memory response with possible implications in serodiagnosis and TB vaccine development.

MCherry-expressing Mycobacterium tuberculosis gives insight into host cell tropism and immune pressure at the single bacterium level in macaques with tuberculosis

Abstract Tuberculosis is caused by Mycobacterium tuberculosis (Mtb) and is associated with granuloma formation in the lungs and other tissues. These lesions can limit bacterial replication but also contain host cells for Mtb and support bacterial persistence and dissemination in poorly controlled disease. Macrophages are often viewed as primary host cells for Mtb but work in mice demonstrates that other cell types host Mtb in acute infection. Less is known about host cell tropism in human TB, especially in the early stages of infection but these early interactions may have implications for later stages of disease. To identify which cells are infected in early and established TB, we infected two cynomolgus macaques with an mCherry-expressing Mtb strain and used flow cytometry and microscopy to identify and phenotype the infected cells at four- and ten-weeks post infection. This approach also allowed us to compare bacterial mCherry expression and size to determine if granulomas exert different levels of pressure on Mtb that vary by timepoint or lesion type. We found that granulomas contained numerous Mtb-infected neutrophils and macrophages at four weeks post infection but fewer infected neutrophils at ten weeks post infection. When the fluorescence and size of individual bacteria were compared across granulomas and timepoints, we found that bacteria in high Mtb burden granulomas were more fluorescent than bacteria in granulomas that contained fewer bacteria, and the bacteria in the four-week granulomas were longer, suggesting that early granulomas are better at supporting Mtb growth than granulomas in established disease. These results provide new insights into Mtb-host cell dynamics in a nonhuman primate model that replicates human TB.

New technologies for diagnosing active TB: the VANTDET diagnostic accuracy study

Background Tuberculosis (TB) is a devastating disease for which new diagnostic tests are desperately needed. Objective To validate promising new technologies [namely whole-blood transcriptomics, proteomics, flow cytometry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)] and existing signatures for the detection of active TB in samples obtained from individuals with suspected active TB. Design Four substudies, each of which used samples from the biobank collected as part of the interferon gamma release assay (IGRA) in the Diagnostic Evaluation of Active TB study, which was a prospective cohort of patients recruited with suspected TB. Setting Secondary care. Participants Adults aged ≥ 16 years presenting as inpatients or outpatients at 12 NHS hospital trusts in London, Slough, Oxford, Leicester and Birmingham, with suspected active TB. Interventions New tests using genome-wide gene expression microarray (transcriptomics), surface-enhanced laser desorption ionisation time-of-flight mass spectrometry/liquid chromatography–mass spectrometry (proteomics), flow cytometry or qRT-PCR. Main outcome measures Area under the curve (AUC), sensitivity and specificity were calculated to determine diagnostic accuracy. Positive and negative predictive values were calculated in some cases. A decision tree model was developed to calculate the incremental costs and quality-adjusted life-years of changing from current practice to using the novels tests. Results The project, and four substudies that assessed the previously published signatures, measured each of the new technologies and performed a health economic analysis in which the best-performing tests were evaluated for cost-effectiveness. The diagnostic accuracy of the transcriptomic tests ranged from an AUC of 0.81 to 0.84 for detecting all TB in our cohort. The performance for detecting culture-confirmed TB or pulmonary TB was better than for highly probable TB or extrapulmonary tuberculosis (EPTB), but was not high enough to be clinically useful. None of the previously described serum proteomic signatures for active TB provided good diagnostic accuracy, nor did the candidate rule-out tests. Four out of six previously described cellular immune signatures provided a reasonable level of diagnostic accuracy (AUC = 0.78–0.92) for discriminating all TB from those with other disease and latent TB infection in human immunodeficiency virus-negative TB suspects. Two of these assays may be useful in the IGRA-positive population and can provide high positive predictive value. None of the new tests for TB can be considered cost-effective. Limitations The diagnostic performance of new tests among the HIV-positive population was either underpowered or not sufficiently achieved in each substudy. Conclusions Overall, the diagnostic performance of all previously identified ‘signatures’ of TB was lower than previously reported. This probably reflects the nature of the cohort we used, which includes the harder to diagnose groups, such as culture-unconfirmed TB or EPTB, which were under-represented in previous cohorts. Future work We are yet to achieve our secondary objective of deriving novel signatures of TB using our data sets. This was beyond the scope of this report. We recommend that future studies using these technologies target specific subtypes of TB, specifically those groups for which new diagnostic tests are required. Funding This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a MRC and NIHR partnership.

Abattoir survey of bovine tuberculosis in tanta, centre of the Nile delta, with in silico analysis of gene mutations and protein-protein interactions of the involved mycobacteria.

Bovine tuberculosis is a transboundary disease of high economic and public health burden worldwide. In this study, post-mortem examination of 750 cattle and buffalo in Tanta abattoir, Centre of the Nile Delta, revealed visible TB in 4% of animals and a true prevalence of 6.85% (95% CI: 5.3%-8.9%). Mycobacterial culture, histopathology and RT-PCR targeting all members of M.tuberculosis complex were performed, upon which 85%, 80% and 100% of each tested lesions were confirmed as TB, respectively. Mpb70-targeting PCR was conducted on ten RT-PCR positive samples for sequencing and identified nine Mycobacterium (M.) bovis strains and, interestingly, one M. tuberculosis (Mtb) strain from a buffalo. Bioinformatics tools were used for prediction of mutations, nucleotide polymorphisms, lineages, drug resistance and protein-protein interactions (PPI) of the sequenced strains. The Mtb strain was resistant to rifampicin, isoniazid and streptomycin, and to the best of our knowledge, this is the first report of multidrug resistant (MDR)-Mtb originating from buffaloes. Seven M.bovis strains were resistant to ethambutol and ethionamide. Such resistances were associated with KatG, rpoB, rpsL, embB and ethA genes mutations. Other mutations and nucleotide polymorphisms were also predicted, some are reported for the first time and require experimental work for validation. PPI revealed more interactions than what would be expected for a random set of proteins of similar size and had dense interactions between nodes that are biologically connected, as a group. Two M.bovis strains belonged to BOV AFRI lineage (Spoligotypes BOV 1; BOV 2) and eight strains belonged to East-Asian (Beijing) lineage. In conclusion, visible TB was prevalent in the study area, RT-PCR is the best to confirm the disease, MDR-Mtb is associated with buffalo TB, and mycobacteria of different lineages carry many resistance genes to chemotherapeutic agents used in treatment of human TB constituting a major public health risk.

Spatial and temporal analyses of culls due to bovine tuberculosis in slaughterhouses of Minas Gerais state, Brazil, 2008 to 2012

ABSTRACT: The application of spatial and temporal analysis in epidemiology aims to understand the causes and consequences of spatial and temporal heterogeneity in studies of infectious diseases. Bovine tuberculosis (bTB) is a chronic and progressive infectious disease caused by Mycobacterium bovis and is an important zoonosis worldwide. The aim of this study was to conduct spatial and temporal analyses of a secondary database of bTB-positive cases registered by the Federal Inspection Service in two slaughterhouses from the West of Minas mesoregion of Minas Gerais state, Brazil, from 2008 to 2012 and to suggest its use in epidemiological surveillance. The culled cattle with bTB macroscopic lesions during post mortem inspection were considered positive in this study. The data used were the positive cases registered on the “Sistema de Informações Gerenciais do Serviço de Inspeção Federal” (SIG/SIF - Management Information System of the Federal Inspection Service) of the “Ministério da Agricultura, Pecuária e Abastecimento” (MAPA - Ministry of Agriculture, Livestock and Supplies), the number of animals slaughtered monthly and their municipalities of origin, per slaughterhouse. The prevalence (%) of cases per cattle slaughtered was calculated, and the relationship between these cases of bTB and cases of human tuberculosis (hTB) and the GDP per capita of the mesoregions comprising the municipalities surveyed was evaluated. The prevalence was 1,030 cases of bTB (0.28%). The lowest case numbers and prevalence rates were obtained in 2010, while the highest rates were observed in 2011. Slaughterhouse 1 showed stationarity, while Slaughterhouse 2 showed an increasing annual trend for data since October 2009. A statistical correlation between the numbers of cases of bovine and human TB (p=0.006, r=0.148) was observed. The prevalence for Slaughterhouses 1 and 2 were not correlated (p&gt;0.05). The municipalities of origin demonstrated randomness in their spatial distribution. SIG/SIF-MAPA may be used for the analysis of spatial and temporal distribution to contribute to the monitoring of animal health services with information on the prevalence of culling for bTB in the West of Minas, South/Southwest of Minas, Campo das Vertentes and Zona da Mata mesoregions of Minas Gerais, Brazil.