Dual oxidase 1 is dispensable during Mycobacterium tuberculosis infection in mice.

Introduction: Mycobacterium tuberculosis (Mtb) is the primary cause of human tuberculosis (TB) and is currently the second most common cause of death due to a singleinfectious agent. The first line of defense against airborne pathogens, including Mtb, is the respiratory epithelium. One of the innate defenses used by respiratory epithelial cells to prevent microbial infection is an oxidative antimicrobial system consisting of the proteins, lactoperoxidase (LPO) and Dual oxidase 1 (Duox1), the thiocyanate anion (SCN-) and hydrogen peroxide (H2O2), which together lead to the generation of antimicrobial hypothiocyanite (OSCN-) in the airway lumen. OSCN- kills bacteria and viruses in vitro, but the role of this Duox1-based system in bacterial infections in vivo remains largely unknown. The goal of this study was to assess whether Duox1 contributes to the immune response against the unique respiratory pathogen, Mtb. Methods: Duox1-deficient (Duox1 KO) and wild-type (WT) mice were infected with Mtb aerosols and bacterial titers, lung pathology, cytokines and immune cell recruitment were assessed. Results and discussion: Mtb titers in the lung, spleen and liver were not different 30 days after infection between WT and Duox1 KO mice. Duox1 did not affect lung histology assessed at days 0, 30, and 90 post-Mtb infection. Mtb-infected Duox1 KO animals exhibited enhanced production of certain cytokines and chemokines in the airway; however, this response was not associated with significantly higher numbers of macrophages or neutrophils in the lung. B cell numbers were lower, while apoptosis was higher in the pulmonary lesions of Mtb-infected Duox1 KO mice compared to infected WT animals. Taken together, these data demonstrate that while Duox1 might influence leukocyte recruitment to inflammatory cell aggregates, Duox1 is dispensable for the overall clinical course of Mtb lung infection in a mouse model.

Ferrets as a model for tuberculosis transmission.

Even with the COVID-19 pandemic, tuberculosis remains a leading cause of human death due to a single infectious agent. Until successfully treated, infected individuals may continue to transmit Mycobacterium tuberculosis bacilli to contacts. As with other respiratory pathogens, such as SARS-CoV-2, modeling the process of person-to-person transmission will inform efforts to develop vaccines and therapies that specifically impede disease transmission. The ferret (Mustela furo), a relatively inexpensive, small animal has been successfully employed to model transmissibility, pathogenicity, and tropism of influenza and other respiratory disease agents. Ferrets can become naturally infected with Mycobacterium bovis and are closely related to badgers, well known in Great Britain and elsewhere as a natural transmission vehicle for bovine tuberculosis. Herein, we report results of a study demonstrating that within 7 weeks of intratracheal infection with a high dose (>5 x 103 CFU) of M. tuberculosis bacilli, ferrets develop clinical signs and pathological features similar to acute disease reported in larger animals, and ferrets infected with very-high doses (>5 x 104 CFU) develop severe signs within two to four weeks, with loss of body weight as high as 30%. Natural transmission of this pathogen was also examined. Acutely-infected ferrets transmitted M. tuberculosis bacilli to co-housed naïve sentinels; most of the sentinels tested positive for M. tuberculosis in nasal washes, while several developed variable disease symptomologies similar to those reported for humans exposed to an active tuberculosis patient in a closed setting. Transmission was more efficient when the transmitting animal had a well-established acute infection. The findings support further assessment of this model system for tuberculosis transmission including the testing of prevention measures and vaccine efficacy.

Genetic Diversity of Mycobacterium tuberculosis Isolates From an Amerindian Population in Chiapas, México.

This is the first report of the genetic diversity of the Mycobacterium tuberculosis complex isolates found in a Mexican-Amerindian setting. In this study, we analyzed isolates collected from the Highlands region of Chiapas, Mexico, by using spoligotyping and whole-genome sequencing analyses. Seventy-three M. tuberculosis isolates were analyzed initially by spoligotyping; no new spoligotypes were identified. Nineteen percent of the isolates were identified as SIT53 (T1) (n = 14), followed by SIT42 (14%, n = 10, LAM9) and SIT119 (11%; n = 8, X1). SIT53, SIT42, and orphan isolates (16.4%, n = 12) constituted about 50% of the isolates studied and were subjected to whole-genome sequencing (WGS) analysis. Most SIT53 (10/12) isolates belonged to the Euro-American sub-lineage 4.8. Most SIT42 isolates (4/7) as .well as most orphan isolates (5/8) belonged to the lineage 4.3.3 LAM group. By comparing the single-nucleotide polymorphism (SNP) patterns of the SIT53 isolates, we found one clone (<7 SNPs) and four clustered isolates (<15 SNPs). In isolates from the SIT42 and orphan groups, we did not find any clones or clusters. This work demonstrates the success of sub-lineage 4.8 to predominate in Mexico and confirms the dominion of sub-lineage 4.3.3 in Central and South America.

CtpB Facilitates Mycobacterium tuberculosis Growth in Copper-Limited Niches.

Copper is required for aerobic respiration by Mycobacterium tuberculosis and its human host, but this essential element is toxic in abundance. Copper nutritional immunity refers to host processes that modulate levels of free copper to alternately starve and intoxicate invading microbes. Bacteria engulfed by macrophages are initially contained within copper-limited phagosomes, which fuse with ATP7A vesicles that pump in toxic levels of copper. In this report, we examine how CtpB, a P-type ATPase in M. tuberculosis, aids in response to nutritional immunity. In vitro, the induced expression of ctpB in copper-replete medium inhibited mycobacterial growth, while deletion of the gene impaired growth only in copper-starved medium and within copper-limited host cells, suggesting a role for CtpB in copper acquisition or export to the copper-dependent respiration supercomplex. Unexpectedly, the absence of ctpB resulted in hypervirulence in the DBA/2 mouse infection model. As ctpB null strains exhibit diminished growth only in copper-starved conditions, reduced copper transport may have enabled the mutant to acquire a "Goldilocks" amount of the metal during transit through copper-intoxicating environments within this model system. This work reveals CtpB as a component of the M. tuberculosis toolkit to counter host nutritional immunity and underscores the importance of elucidating copper-uptake mechanisms in pathogenic mycobacteria.

Characterization of the Proportion of Clustered Tuberculosis Cases in Guatemala: Insights from a Molecular Epidemiology Study, 2010-2014.

There is little information about the amount of recent tuberculosis transmission in low-income settings. Genetic clustering can help identify ongoing transmission events. A retrospective observational study was performed on Mycobacterium tuberculosis isolates from persons living with HIV (PLHIV) and HIV-seronegative participants who submitted samples to a referral tuberculosis laboratory in Guatemala City, Guatemala from 2010 to 2014. Genotyping results were classified according to the international spoligotyping database, SITVIT2. Spoligotype patterns were categorized as clustered or nonclustered depending on their genotype. The proportion of clustering and the index of recent transmission index (RTIn-1) were estimated. In the RTIn-1 method, clustered cases represent recent transmission, whereas nonclustered cases represent reactivation of older tuberculosis infections. As a secondary aim, the potential risk factors associated with clustering in isolates from the subset of participants living with HIV were explored. From 2010 to 2014, a total of 479 study participants were confirmed as culture-positive tuberculosis cases. Among the 400 available isolates, 71 spoligotype patterns were identified. Overall, the most frequent spoligotyping families were Latin American-Mediterranean (LAM) (39%), followed by T (22%) and Haarlem (14%). Out of the 400 isolates, 365 were grouped in 36 clusters (range of cluster size: 2-92). Thus, the proportion of clustering was 91% and the RTIn-1 was 82%. Among PLHIV, pulmonary tuberculosis was associated with clustering (OR = 4.3, 95% CI 1.0-17.7). Our findings suggest high levels of ongoing transmission of M. tuberculosis in Guatemala as revealed by the high proportion of isolates falling into genomic clusters.

Use of a Ferret Model to Test Efficacy and Immunogenicity of Live Attenuated Mycobacterium avium Subspecies paratuberculosis Vaccines.

Native hosts for the bacterial agent that causes Johne's disease are ruminants, which include cattle, sheep and goats among others. These large animals are often too costly to be used in testing experimental vaccines. In this chapter, we provide detailed methods to use an inexpensive and more manageable animal host, the ferret, to test efficacy and immunogenicity of live-attenuated Mycobacterium avium subspecies paratuberculosis (MAP) mutant strains prior to consideration as vaccine candidates.

Assessing a transmission network of Mycobacterium tuberculosis in an African city using single nucleotide polymorphism threshold analysis.

Tuberculosis (TB) is the leading cause of death in humans by a single infectious agent worldwide with approximately two billion humans latently infected with the bacterium Mycobacterium tuberculosis. Currently, the accepted method for controlling the disease is Tuberculosis Directly Observed Treatment Shortcourse (TB-DOTS). This program is not preventative and individuals may transmit disease before diagnosis, thus better understanding of disease transmission is essential. Using whole-genome sequencing and single nucleotide polymorphism analysis, we analyzed genomes of 145 M. tuberculosis clinical isolates from active TB cases from the Rubaga Division of Kampala, Uganda. We established that these isolates grouped into M. tuberculosis complex (MTBC) lineages 1, 2, 3, and 4, with the most isolates grouping into lineage 4. Possible transmission pairs containing ≤12 SNPs were identified in lineages 1, 3, and 4 with the prevailing transmission in lineages 3 and 4. Furthermore, investigating DNA codon changes as a result of specific SNPs in prominent virulence genes including plcA and plcB could indicate potentially important modifications in protein function. Incorporating this analysis with corresponding epidemiological data may provide a blueprint for the integration of public health interventions to decrease TB transmission in a region.

A Role for Mycobacterium tuberculosis Sigma Factor C in Copper Nutritional Immunity.

Sigma factor C (SigC) contributes to Mycobacterium tuberculosis virulence in various animal models, but the stress response coordinated by this transcription factor was undefined. The results presented here indicate that SigC prevents copper starvation. Whole genome expression studies demonstrate short-term (4-h) induction of sigC, controlled from a tetracycline-inducible promoter, upregulates ctpB and genes in the nonribosomal peptide synthase (nrp) operon. These genes are expressed at higher levels after 48-h sigC induction, but also elevated are genes encoding copper-responsive regulator RicR and RicR-regulated copper toxicity response operon genes rv0846-rv0850, suggesting prolonged sigC induction results in excessive copper uptake. No growth and global transcriptional differences are observed between a sigC null mutant relative to its parent strain in 7H9 medium. In a copper-deficient medium, however, growth of the sigC deletion strain lags the parent, and 40 genes (including those in the nrp operon) are differentially expressed. Copper supplementation reverses the growth defect and silences most transcriptional differences. Together, these data support SigC as a transcriptional regulator of copper acquisition when the metal is scarce. Attenuation of sigC mutants in severe combined immunodeficient mice is consistent with an inability to overcome innate host defenses that sequester copper ions to deprive invading microbes of this essential micronutrient.

Low-Dose Exposure to Ganglioside-Mimicking Bacteria Tolerizes Human Macrophages to Guillain-Barré Syndrome-Associated Antigens.

Early in life, commensal bacteria play a major role in immune development, helping to guide the host response toward harmful stimuli while tolerating harmless antigens to prevent autoimmunity. Guillain-Barré syndrome (GBS) is an autoimmune disease caused by errant immune attack of antibody-bound ganglioside receptors on host nerve cells, resulting in paralysis. Lipooligosaccharides enveloping the prevalent enteric pathogen, Campylobacter jejuni, frequently mimic human gangliosides and can trigger GBS by stimulating the autoimmune response. In low- to middle-income countries, young children are consistently exposed to C. jejuni, and it is not known if this impacts GBS susceptibility later in life. Using a macrophage model, we examined the effect of training these cells with low doses of ganglioside-mimicking bacteria prior to challenge with GBS-associated antigens. This training caused decreased production of proinflammatory cytokines, suggesting tolerance induction. We then screened Campylobacter isolates from 154 infant fecal samples for GM1 ganglioside mimicry, finding that 23.4% of strains from both symptomatic and asymptomatic infants displayed GM1-like structures. Training macrophages with one of these asymptomatic carrier isolates also induced tolerance against GBS-associated antigens, supporting that children can be exposed to the tolerizing antigen early in life. RNA interference of Toll-like receptor 2 (TLR2) and TLR4 suggests that these receptors are not involved in tolerance associated with decreases in tumor necrosis factor (TNF), interleukin-6 (IL-6), or IL-1ß levels. The results of this study suggest that exposure to ganglioside-mimicking bacteria early in life occurs naturally and impacts host susceptibility to GBS development. IMPORTANCE In this study, we demonstrated that it is possible to tolerize immune cells to potentially dampen the autoreactive proinflammatory immune response against Guillain-Barré syndrome (GBS)-associated antigens. The innate immune response functions to arm the host against bacterial attack, but it can be tricked into recognizing the host's own cells when infectious bacteria display sugar structures that mimic human glycans. It is this errant response that leads to the autoimmunity and paralysis associated with GBS. By presenting immune cells with small amounts of the bacterial glycan mimic, we were able to suppress the proinflammatory immune response upon subsequent high exposure to glycan-mimicking bacteria. This suggests that individuals who have already been exposed to the glycan mimics in small amounts are less sensitive to autoimmune reactions against these glycans, and this could be a factor in determining susceptibility to GBS.

Excess Risk of Tuberculosis Infection Among Extra-household Contacts of Tuberculosis Cases in an African City.

BACKGROUND: Although households of tuberculosis (TB) cases represent a setting for intense transmission of Mycobacterium tuberculosis, household exposure accounts for <20% of transmission within a community. The aim of this study was to estimate excess risk of M. tuberculosis infection among household and extra-household contacts of index cases. METHODS: We performed a cross-sectional study in Kampala, Uganda, to delineate social networks of TB cases and matched controls without TB. We estimated the age-stratified prevalence difference of TB infection between case and control networks, partitioned as household and extra-household contacts. RESULTS: We enrolled 123 index cases, 124 index controls, and 2415 first-degree network contacts. The prevalence of infection was highest among household contacts of cases (61.5%), lowest among household contacts of controls (25.2%), and intermediary among extra-household TB contacts (44.9%) and extra-household control contacts (41.2%). The age-adjusted prevalence difference between extra-household contacts of cases and their controls was 5.4%. The prevalence of infection was similar among the majority of extra-household case contacts and corresponding controls (47%). CONCLUSIONS: Most first-degree social network members of TB cases do not have adequate contact with the index case to experience additional risk for infection, but appear instead to acquire infection through unrecognized exposures with infectious cases in the community.

Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication.

Salmonellosis and listeriosis together accounted for more than one third of foodborne illnesses in the United States and almost half the hospitalizations for gastrointestinal diseases in 2018 while tuberculosis afflicted over 10 million people worldwide causing almost 2 million deaths. Regardless of the intrinsic virulence differences among Listeria monocytogenes, Salmonella enterica and Mycobacterium tuberculosis, these intracellular pathogens share the ability to survive and persist inside the macrophage and other cells and thrive in iron rich environments. Interferon-gamma (IFN-γ) is a central cytokine in host defense against intracellular pathogens and has been shown to promote iron export in macrophages. We hypothesize that IFN-γ decreases iron availability to intracellular pathogens consequently limiting replication in these cells. In this study, we show that IFN-γ regulates the expression of iron-related proteins hepcidin, ferroportin, and ferritin to induce iron export from macrophages. Listeria monocytogenes, S. enterica, and M. tuberculosis infections significantly induce iron sequestration in human macrophages. In contrast, IFN-γ significantly reduces hepcidin secretion in S. enterica and M. tuberculosis infected macrophages. Similarly, IFN-γ-activated macrophages express higher ferroportin levels than untreated controls even after infection with L. monocytogenes bacilli; bacterial infection greatly down-regulates ferroportin expression. Collectively, IFN-γ significantly inhibits pathogen-associated intracellular iron sequestration in macrophages and consequently retards the growth of intracellular bacterial pathogens by decreasing iron availability.

BCG vaccination for bovine tuberculosis; conclusions from the Jerusalem One Health workshop.

The global burden of bovine tuberculosis (bTB) remains poorly characterized, with spill-over impacts on multiple species. The "One Health" concept is especially relevant given the bidirectional risk of cattle infecting humans with Mycobacterium bovis and humans infecting cattle with Mycobacterium tuberculosis. "Test and cull" is the traditional bTB control method, but the strategy may not be economically feasible or culturally acceptable where cattle are highly prized or their killing is a religious taboo; it is also less effective when there are wildlife reservoirs of infection. Vaccination with M. bovis bacille Calmette-Guerin (BCG) provides protection against bTB, but its use in animals has been limited. The Jerusalem One Health workshop considered key bTB knowledge gaps and innovative solutions. Knowledge gaps identified included (a) the poorly quantified prevalence of M. bovis infection and disease in cattle, domestic camelids and human populations in developing countries, (b) the absence of alternatives to a "test and cull" strategy in settings where the killing of infected animals is culturally or economically unacceptable, or where affected species are protected and (c) an understanding of the induction of mucosal immunity against bTB. We summarize discussions on the use of BCG vaccination in domestic animals and wildlife and list potential projects to address the knowledge gaps identified.

Evaluation of a temperature-restricted, mucosal tuberculosis vaccine in guinea pigs.

Tuberculosis (TB) is currently the leading cause of death in humans by a single infectious agent, Mycobacterium tuberculosis. The Bacillus Calmette-Guérin (BCG) vaccine prevents pulmonary TB with variable efficacy, but can cause life-threatening systemic infection in HIV-infected infants. In this study, TBvac85, a derivative of Mycobacterium shottsii expressing M. tuberculosis Antigen 85B, was examined as a safer alternative to BCG. Intranasal vaccination of guinea pigs with TBvac85, a naturally temperature-restricted species, resulted in serum Ag85B-specific IgG antibodies. Delivery of the vaccine by this route also induced protection equivalent to intradermal BCG based on organ bacterial burdens and lung pathology six weeks after aerosol challenge with M. tuberculosis strain Erdman. These results support the potential of TBvac85 as the basis of an effective TB vaccine. Next-generation derivatives expressing multiple M. tuberculosis immunogens are in development.

Role of the hepcidin-ferroportin axis in pathogen-mediated intracellular iron sequestration in human phagocytic cells.

Upon infection, pathogen and host compete for the same iron pool, because this trace metal is a crucial micronutrient for all living cells. Iron dysregulation in the host is strongly associated with poor outcomes in several infectious diseases, including tuberculosis, AIDS, and malaria, and inefficient iron scavenging by pathogens severely affects their virulence. Hepcidin is the master regulator of iron homeostasis in vertebrates, responsible for diminishing iron export from macrophages during iron overload or infection. Hepcidin regulation in hepatocytes is well characterized and mostly dependent on interleukin-6 signaling during inflammation, although in myeloid cells, hepcidin induction and the mechanisms leading to intracellular iron regulation remain elusive. Here we show that activation of different Toll-like receptors (TLRs) by their respective ligands leads to increased iron sequestration in macrophages. By measuring the transcriptional levels of iron-related proteins (eg, hepcidin, ferroportin, and ferritin), we observed that TLR signaling can induce intracellular iron sequestration in macrophages through 2 independent but redundant mechanisms. Interestingly, TLR2 ligands or infection with Listeria monocytogenes lead to direct ferroportin transcriptional downregulation, whereas TLR4 ligands, such as lipopolysaccharide, induce hepcidin expression. Infection with Mycobacterium bovis Bacillus Calmette-Guerin promotes intracellular iron sequestration through both hepcidin upregulation and ferroportin downregulation. This is the first study in which TLR1-9-mediated iron homeostasis in human macrophages was evaluated, and the outcome of this study elucidates the mechanism of iron dysregulation in macrophages during infection.

Heparin inhibits intracellular Mycobacterium tuberculosis bacterial replication by reducing iron levels in human macrophages.

Iron is a crucial micronutrient for both mammals and their associated pathogens, and extensive literature has shown that Mycobacterium tuberculosis (Mtb) bacilli inhibited from acquiring iron from the host are severely attenuated. In contrast, increased dietary iron concentrations or patients with hemochromatosis have long been associated with a more severe tuberculosis (TB) disease outcome. We have observed that upon macrophage infection, Mtb bacilli strongly promote intracellular iron sequestration, both through increased expression of hepcidin, a key mammalian iron regulatory protein, and downregulation of the iron exporter protein, ferroportin. Heparin is a highly sulfated glycosaminoglycan released by mast cells and basophils at sites of tissue injury. During Mtb infection, heparin alters intracellular trafficking in alveolar epithelial cells and decreases extrapulmonary dissemination but recently, heparin also has been reported to inhibit hepcidin expression in hepatocytes, decreasing intracellular iron availability. In this report, we demonstrate that heparin significantly reduces hepcidin expression in macrophages infected with Mtb bacilli. Heparin-treated macrophages have higher ferroportin expression compared to untreated macrophages, promoting iron export and decreasing iron availability to intracellular bacilli. Thus, here we describe a novel immunomodulatory effect and potential therapeutic role for heparin against mycobacterial infection in human macrophages.

Effectiveness of WHO's pragmatic screening algorithm for child contacts of tuberculosis cases in resource-constrained settings: a prospective cohort study in Uganda.

BACKGROUND: Tuberculosis is a leading cause of global childhood mortality; however, interventions to detect undiagnosed tuberculosis in children are underused. Child contact tracing has been widely recommended but poorly implemented in resource-constrained settings. WHO has proposed a pragmatic screening approach for managing child contacts. We assessed the effectiveness of this screening approach and alternative symptom-based algorithms in identifying secondary tuberculosis in a prospectively followed cohort of Ugandan child contacts. METHODS: We identified index patients aged at least 18 years with microbiologically confirmed pulmonary tuberculosis at Old Mulago Hospital (Kampala, Uganda) between Oct 1, 1995, and Dec 31, 2008. Households of index patients were visited by fieldworkers within 2 weeks of diagnosis. Coprevalent and incident tuberculosis were assessed in household contacts through clinical, radiographical, and microbiological examinations for 2 years. Disease rates were compared among children younger than 16 years with and without symptoms included in the WHO pragmatic guideline (presence of haemoptysis, fever, chronic cough, weight loss, night sweats, and poor appetite). Symptoms could be of any duration, except cough (>21 days) and fever (>14 days). A modified WHO decision-tree designed to detect high-risk asymptomatic child contacts was also assessed, in which all asymptomatic contacts were classified as high risk (children younger than 3 years or immunocompromised [HIV-infected]) or low risk (aged 3 years or older and immunocompetent [HIV-negative]). We also assessed a more restrictive algorithm (ie, assessing only children with presence of chronic cough and one other tuberculosis-related symptom). FINDINGS: Of 1718 household child contacts, 126 (7%) had coprevalent tuberculosis and 24 (1%) developed incident tuberculosis, diagnosed over the 2-year study period. Of these 150 cases of tuberculosis, 95 (63%) were microbiologically confirmed with a positive sputum culture. Using the WHO approach, 364 (21%) of 1718 child contacts had at least one tuberculosis-related symptom and 85 (23%) were identified as having coprevalent tuberculosis, 67% of all coprevalent cases detected (diagnostic odds ratio 9·8, 95% CI 6·8-14·5; p<0·0001). 1354 (79%) of 1718 child contacts had no symptoms, of whom 41 (3%) had coprevalent tuberculosis. The WHO approach was effective in contacts younger than 5 years: 70 (33%) of 211 symptomatic contacts had coprevalent disease compared with 23 (6%) of 367 asymptomatic contacts (p<0·0001). This approach was also effective in contacts aged 5 years and older: 15 (10%) of 153 symptomatic contacts had coprevalent disease compared with 18 (2%) of 987 asymptomatic contacts (p<0·0001). More coprevalent disease was detected in child contacts recommended for screening when the study population was restricted by HIV-serostatus (11 [48%] of 23 symptomatic HIV-seropositive child contacts vs two [7%] of 31 asymptomatic HIV-seropositive child contacts) or to only culture-confirmed cases (47 [13%] culture confirmed cases of 364 symptomatic child contacts vs 29 [2%] culture confirmed cases of 1354 asymptomatic child contacts). In the modified algorithm, high-risk asymptomatic child contacts were at increased risk for coprevalent disease versus low-risk asymptomatic contacts (14 [6%] of 224 vs 27 [2%] of 1130; p=0·0021). The presence of tuberculosis infection did not predict incident disease in either symptomatic or asymptomatic child contacts: in symptomatic contacts, eight (5%) of 169 infected contacts and six (5%) of 111 uninfected contacts developed incident tuberculosis (p=0·80). Among asymptomatic contacts, incident tuberculosis occurred in six (<1%) of 795 contacts infected at baseline versus four (<1%) of 518 contacts uninfected at baseline, respectively (p=1·00). INTERPRETATION: WHO's pragmatic, symptom-based algorithm was an effective case-finding tool, especially in children younger than 5 years. A modified decision-tree identified 6% of asymptomatic child contacts at high risk for subclinical disease. Increasing the feasibility of child-contact tracing using these approaches should be encouraged to decrease tuberculosis-related paediatric mortality in high-burden settings, but this should be partnered with increasing access to microbiological point-of-care testing. FUNDING: National Institutes of Health, Tuberculosis Research Unit, AIDS International Training and Research Program of the Fogarty International Center, and the Center for AIDS Research.

Detection of anti-HspX antibodies and HspX protein in patient sera for the identification of recent latent infection by Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a pathogen causing tuberculosis (TB) a spectrum of disease including acute and asymptomatic latent stages. Identifying and treating latently-infected patients constitutes one of the most important impediments to TB control efforts. Those individuals can remain undiagnosed for decades serving as potential reservoirs for disease reactivation. Tests for the accurate diagnosis of latent infection currently are unavailable. HspX protein (α-crystallin), encoded by Rv2031c gene, is produced in vitro by M. tuberculosis during stationary growth phase and hypoxic or acidic culture conditions. In this study, using standard, and Luminex xMAP® bead capture ELISA, respectively, we report on detection of anti-HspX IgG and IgM antibodies and HspX protein in sera from acute and latent TB patients. For the antibody screen, levels of IgG and IgM antibodies were similar between non-infected and active TB patients; however, individuals classified into the group with latent TB showed higher values of anti-HspX IgM (p = 0.003) compared to active TB patients. Using the bead capture antigen detection assay, HspX protein was detected in sera from 56.5% of putative latent cases (p< 0.050) compared to the background median with an average of 9,900 pg/ml and a range of 1,000 to 36,000 pg/ml. Thus, presence of anti-HspX IgM antibodies and HspX protein in sera may be markers of latent TB.

An acidic sphingomyelinase Type C activity from Mycobacterium tuberculosis.

Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Sphingolipids are recognized as diverse and dynamic regulators of a multitude of cellular processes mediating cell cycle control, differentiation, stress response, cell migration, adhesion, and apoptosis. Bacterial SMases are virulence factors for several species of pathogens. Whole cell extracts of Mycobacterium tuberculosis strains H37Rv and CDC1551 were assayed using [N-methyl-(14)C]-sphingomyelin as substrate. Acidic Zn(2+)-dependent SMase activity was identified in both strains. Peak SMase activity was observed at pH 5.5. Interestingly, overall SMase activity levels from CDC1551 extracts are approximately 1/3 of those of H37Rv. The presence of exogenous SMase produced by M. tuberculosis during infection may interfere with the normal host inflammatory response thus allowing the establishment of infection and disease development. This Type C activity is different from previously identified M. tuberculosis SMases. Defining the biochemical characteristics of M. tuberculosis SMases helps to elucidate the roles that these enzymes play during infection and disease.

Actividad de una esfingomielinasa ácida tipo C producida por Mycobacterium tuberculosis / An acidic sphingomyelinase Type C activity from Mycobacterium tuberculosis

Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Sphingolipids are recognized as diverse and dynamic regulators of a multitude of cellular processes mediating cell cycle control, differentiation, stress response, cell migration, adhesion, and apoptosis. Bacterial SMases are virulence factors for several species of pathogens. Whole cell extracts of Mycobacterium tuberculosis strains H37Rv and CDC1551 were assayed using [N-methyl-14C]-sphingomyelin as substrate. Acidic Zn2+-dependent SMase activity was identified in both strains. Peak SMase activity was observed at pH 5.5. Interestingly, overall SMase activity levels from CDC1551 extracts are approximately 1/3 of those of H37Rv. The presence of exogenous SMase produced by M. tuberculosis during infection may interfere with the normal host inflammatory response thus allowing the establishment of infection and disease development. This Type C activity is different from previously identified M. tuberculosis SMases. Defining the biochemical characteristics of M. tuberculosis SMases helps to elucidate the roles that these enzymes play during infection and disease.

Las esfingomielinasas (SMasas) catalizan la hidrólisis de esfingomielina a ceramida y fosforilcolina. Los esfingolípidos son reconocidos como reguladores diversos y dinámicos de una multitud de procesos celulares que median en el control del ciclo celular, la diferenciación, la respuesta al estrés, la migración celular, la adhesión y la apoptosis. Las esfingomielinasas bacterianas son factores de virulencia reconocidos en varias especies de patógenos. En este trabajo se analizaron los extractos de células enteras de las cepas de Mycobacterium tuberculosis H37Rv y CDC1551 utilizando [N-metil-14C]-esfingomielina como sustrato. Se identificó actividad de SMasa-ácida dependiente de zinc en ambas cepas. La actividad máxima se observó a pH 5.5. Curiosamente, los niveles de actividad de SMasa generados a partir de extractos de la cepa CDC1551 son aproximadamente un tercio de los de la cepa H37Rv. La presencia de una SMasa exógena producida por M. tuberculosis durante la infección puede interferir con la respuesta inflamatoria del huésped, permitiendo así el establecimiento de la infección y el desarrollo de la enfermedad. Esta actividad tipo C es distinta de las actividades previamente reportadas para M. tuberculosis. Definir las características bioquímicas de las esfingomielinasas de M. tuberculosis ayudará a dilucidar el papel que desempeñan estas enzimas durante la infección y la enfermedad.