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1.  Effect of Non-tuberculous Mycobacteria on Host Biomarkers Potentially Relevant for Tuberculosis Management 
Non-tuberculous mycobacteria (NTM) are different from Mycobacterium tuberculosis (MTB) both in their ubiquitous environmental distribution and in their reduced capacity to cause disease. While often neglected in favour of other infectious diseases, NTM may interfere with important aspects of TB control and management, namely the efficacy of new anti-tuberculosis (TB) vaccines; the immuno-diagnostic Tuberculin skin test (TST) and QuantiFERON TB Gold In Tube assay (QFTGIT); and immune biomarkers explored for their diagnostic and/or predictive potential. Our objective was therefore to explore host immune biomarkers in children who had NTM isolated from respiratory and/or gastric specimens.
Methodology and Principle Findings
The present study was nested within a prospective cohort study of BCG-vaccinated neonates in Southern India. In this setting, immune biomarkers from peripheral blood were analyzed in 210 children aged <3 years evaluated for TB using dual-colour-Reverse-Transcriptase-Multiple-Ligation-dependent-Probe-Amplification (dcRT-MLPA) and Bio-Plex assays. The children were classified based on clinical examination, chest X-rays and mycobacterial culture reports as either: 1) TB disease, 2) NTM present and 3) controls. The study shows a down-regulation of RAB33A (p<0.001) and up-regulation of TGFβ1, IL-2 and IL-6 (all p<0.05) in children with TB disease, and that RAB33A, TGFBR2 and IL-10 (all p<0.05) were differentially expressed in children with NTM present when compared to children that were culture negative for MTB and NTM (controls).
Conclusions and Significance
Carriage of NTM may reduce the specificity of future diagnostic and predictive immune biomarkers relevant to TB management.
Author Summary
Non-tuberculous mycobacteria (NTM) are a ubiquitous group of mycobacteria found in the environment. They are opportunistic pathogens causing human disease, especially in immunocompromised individuals. Differentiation between NTM infection and tuberculosis (TB) can be difficult. Data on incidence of NTM in TB endemic countries is limited due to resource intensive methods required for identification and a considerable workload due to other diseases. The present study was based on children investigated for TB and classified according to chest X-rays and mycobacterial culture reports. We explored host immune biomarkers which are potentially relevant to TB management, in children with confirmed NTM exposure. The findings from the present study suggest that NTM exposure modulates TB-relevant immune biomarkers in the host by eliciting some of the same immune responses as MTB infection. This is may be of importance when evaluating immunological correlates of protection in the setting of TB vaccine trials and potential TB diagnostic biomarkers.
PMCID: PMC4199571  PMID: 25329719
2.  Combination of Cytokine Responses Indicative of Latent TB and Active TB in Malawian Adults 
PLoS ONE  2013;8(11):e79742.
An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease.
Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested.
Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses.
CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB.
PMCID: PMC3832606  PMID: 24260295
3.  Association of dietary fat intakes with risk of esophageal and gastric cancer in the NIH-AARP Diet and Health study 
The aim of this study was to investigate whether intakes of total fat and fat subtypes were associated with esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), gastric cardia or gastric non-cardia adenocarcinoma. From 1995–1996, dietary intake data was reported by 494,978 participants of the NIH-AARP cohort. 630 EAC, 215 ESCC, 454 gastric cardia and 501 gastric non-cardia adenocarcinomas accrued to the cohort. Cox proportional hazards regression was used to examine the association between the dietary fat intakes, whilst adjusting for potential confounders. Though apparent associations were observed in energy-adjusted models, multivariate adjustment attenuated results to null (e.g. EAC energy adjusted hazard ratio (HR) and 95% confidence interval (95%CI) 1.66 (1.27–2.18) P for trend <0.01; EAC multivariate adjusted HR (95%CI) 1.17 (0.84–1.64) P for trend=0.58). Similar patterns were also observed for fat subtypes (e.g. EAC saturated fat, energy adjusted HR (95%CI) 1.79 (1.37–2.33) P for trend <0.01; EAC saturated fat, multivariate adjusted HR (95%CI) 1.27 (0.91–1.78) P for trend=0.28). However, in multivariate models an inverse association for polyunsaturated fat (continuous) was seen for EAC in subjects with a body mass index (BMI) in the normal range (18.5–<25 kg/m2) (HR (95%CI) 0.76 (0.63–0.92)), that was not present in overweight subjects (HR (95%CI) 1.04 (0.96–1.14)), or in unstratified analysis (HR (95%CI) 0.97 (0.90–1.05)). P for interaction=0.02. Overall, we found null associations between the dietary fat intakes with esophageal or gastric cancer risk; though a protective effect of polyunsaturated fat intake was seen for EAC in subjects with a normal BMI.
PMCID: PMC3346853  PMID: 22116732
cohort; dietary fat; esophageal neoplasms; stomach neoplasms; prospective
4.  Two-Step Tuberculin Skin Testing in School-Going Adolescents with Initial 0-4 Millimeter Responses in a High Tuberculosis Prevalence Setting in South India 
PLoS ONE  2013;8(9):e71470.
The utility of two-step tuberculin skin testing among adolescents in high tuberculosis prevalence settings is not well established.
To determine the proportion and determinants of a 0-4 mm response to an initial standard tuberculin skin test (TST) and evaluating 'boosting' with repeat testing.
Adolescents between 11 and 18 years attending schools/colleges underwent a TST; those with a response of between 0–4 mm had a repeat TST 1-4 weeks later.
Initial TST was done for 6608/6643 participants; 1257 (19%) developed a 0-4 mm response to the initial TST. Younger age and under-nutrition were more likely to be associated with a 0-4 mm response, while the presence of BCG (Bacillus Calmette Guerin) scar and higher socio-economic class were less likely to be associated with a 0-4 mm response. On repeat testing boosting was seen in 13.2% (145/1098; ≥ 6 mm over the initial test) while 4.3% showed boosting using a more conservative cutoff of a repeat TST ≥ 10 mm with an increment of at least 6 mm (47/1098). History of exposure to a tuberculosis (TB) case was associated with enhanced response.
The proportion of adolescents who demonstrated boosting on two-step TST testing in our study was relatively low. As a result repeat testing did not greatly alter the prevalence of TST positivity. However, the two-step TST helps identify individuals who can potentially boost their immune response to a second test, and thus, prevents them from being misclassified as those with newly acquired infection, or tuberculin converters. While two-step tuberculin skin testing may have a limited role in population- level TST surveys, it may be useful where serial tuberculin testing needs to be performed to distinguish those who show an enhanced response or boosters from those who indeed have a new infection, or converters.
PMCID: PMC3765300  PMID: 24039716
5.  Parasitic infection may be associated with discordant responses to QuantiFERON and tuberculin skin test in apparently healthy children and adolescents in a tuberculosis endemic setting, Ethiopia 
BMC Infectious Diseases  2013;13:265.
M. tuberculosis remains one of the world’s deadliest pathogens in part because of its ability to establish persistent, latent infections, which can later reactivate to cause disease. In regions of the globe where disease is endemic, as much as 50% of the population is thought to be latently infected, complicating diagnosis and tuberculosis control. The tools most commonly used for diagnosis of latent M. tuberculosis infection are the tuberculin skin test and the newer interferon-gamma release assays, both of which rely on an antigen-specific memory response as an indicator of infection. It is clear that the two tests, do not always give concordant results, but the factors leading to this are only partially understood.
In this study we examined 245 healthy school children aged from 12 to 20 years from Addis Ababa, a tuberculosis-endemic region, characterised them with regard to response in the tuberculin skin test and QuantIFERON™ test and assessed factors that might contribute to discordant responses.
Although concordance between the tests was generally fair (90% concordance), there was a subset of children who had a positive QuantIFERON™ result but a negative tuberculin skin test. After analysis of multiple parameters the data suggest that discordance was most strongly associated with the presence of parasites in the stool.
Parasitic gut infections are frequent in most regions where M. tuberculosis is endemic. This study, while preliminary, suggests that the tuberculin skin test should be interpreted with caution where this may be the case.
PMCID: PMC3674899  PMID: 23738853
Latent tuberculosis; QuantiFERON; TST; Adolescents; Children; Parasites
6.  Hazardous alcohol consumption is associated with increased levels of B-type natriuretic peptide: evidence from two population-based studies 
European Journal of Epidemiology  2013;28(5):393-404.
Russia has very high mortality from cardiovascular disease (CVD), with evidence that heavy drinking may play a role. To throw further light on this association we have studied the association of alcohol with predictors of CVD risk including B-type natriuretic peptide (BNP). Levels of BNP increase primarily in response to abnormal cardiac chamber wall stretch which can occur both as a result of atherosclerosis as well as due to other types of damage to the myocardium. No previous population-based studies have investigated the association with alcohol. We analysed cross-sectional data on drinking behaviour in 993 men aged 25–60 years from the Izhevsk Family Study 2 (IFS2), conducted in the Russian city of Izhevsk in 2008–2009. Relative to non-drinkers, men who drank hazardously had an odds ratio (OR) of being in the top 20 % of the BNP distribution of 4.66 (95 % CI 2.13, 10.19) adjusted for age, obesity, waist–hip ratio, and smoking. Further adjustment for class of hypertension resulted in only slight attenuation of the effect, suggesting that this effect was not secondary to the influence of alcohol on blood pressure. In contrast hazardous drinking was associated with markedly raised ApoA1 and HDL cholesterol levels, but had little impact on levels of ApoB and LDL cholesterol. Similar but less pronounced associations were found in the Belfast (UK) component of the PRIME study conducted in 1991. These findings suggest that the association of heavy drinking with increased risk of cardiovascular disease may be partly due to alcohol-induced non-atherosclerotic damage to the myocardium.
PMCID: PMC3672507  PMID: 23645505
Alcohol; B-type natriuretic peptide; Russia; Cardiovascular disease
7.  Expression of TNF-Alpha-Dependent Apoptosis-Related Genes in the Peripheral Blood of Malagasy Subjects with Tuberculosis 
PLoS ONE  2013;8(4):e61154.
The majority of Mycobacterium tuberculosis (Mtb) infections remain asymptomatic with only up to 10% progressing to clinical tuberculosis. However, the constituents of the effective “protective immunity” against tuberculosis responsible for containing most infections remain unknown. Evaluating gene transcriptional profiles in tuberculosis clinical cohorts is one approach to understanding the spectrum of tuberculosis progression. It is clear that apoptosis plays a role in the control of tuberculosis but the utility of apoptosis-related genes as surrogate markers of protection against tuberculosis has not been well investigated. To characterize potential surrogate markers that could discriminate different phases of the clinical tuberculosis spectrum, we investigated gene expression of several TNF-alpha dependent apoptotic genes (TNFR1, TNFR2, FLICE, FLIPs) by real-time RT-PCR of peripheral blood cells from cohorts of individuals with active tuberculosis or potential exposure to tuberculosis.
Newly diagnosed tuberculosis patients (n = 23), their close household contacts (n = 80), and community controls (n = 46) were tested at intervals over a period of up to two years. Latent infection or previous Mtb contact was assessed by ELISPOT and TST and complete blood counts were performed during the follow up.
Results showed significant upregulation of FLIPs expression by infected individuals regardless of clinical status at entry to the study. A higher percentage of lymphocytes was found in the infected household contacts that remained healthy. In contrast, in individuals with active TB, a significant upregulation of TNFR2 expression, a significantly higher percentage of monocytes and a significantly decreased lymphocyte count were seen, compared to subjects that remained healthy. Moreover, the household contacts who subsequently developed signs of TB also had a significantly high number of monocytes.
These data suggest tuberculosis may be associated with decreased T-cell survival (perhaps due to apoptosis) while inhibition of apoptosis in monocytes could lead to a relative increase in these cells: a situation predicted to favour Mtb.
PMCID: PMC3625145  PMID: 23593415
8.  A prospective cohort study of obesity and risk of esophageal and gastric adenocarcinoma in the NIH-AARP Diet and Health study 
Gut  2011;61(9):1261-1268.
Incidence of esophageal adenocarcinoma (EAC) has increased rapidly over the past forty years and accumulating evidence suggests that obesity, as measured by body mass index (BMI), is a major risk factor. However, it remains unclear whether abdominal obesity is associated with esophageal and gastric adenocarcinoma.
Cox proportional hazards regression was used to examine associations between overall and abdominal obesity with EAC and gastric adenocarcinoma among 218,854 participants in the prospective NIH-AARP cohort.
253 incident EAC, 191 gastric cardia adenocarcinomas, and 125 gastric non-cardia adenocarcinomas accrued to the cohort. Overall obesity (BMI) was positively associated with EAC and gastric cardia adenocarcinoma risk (highest [≥35 kg/m2] versus referent [18.5–<25 kg/m2]; hazard ratio (HR) 95% confidence interval (95% CI); 2.11 (1.09–4.09) and 3.67 (2.00–6.71), respectively). Waist circumference was also positively associated with EAC and gastric cardia adenocarcinoma risk, (highest versus referent; HR (95% CI) 2.01 (1.35–3.00) and 2.22 (1.43–3.47), respectively), whereas waist-to-hip ratio (WHR) was positively associated with EAC risk only (highest versus referent; HR (95% CI) 1.81 (1.24–2.64)); persisted in patients with normal BMI (18.5–<25 kg/m2). Mutual adjustment of WHR and BMI attenuated both, but did not eliminate the positive associations for either with risk of EAC. In contrast, the majority of the anthropometric variables were not associated with adenocarcinomas of the gastric non-cardia.
Overall obesity was associated with a higher risk of EAC and gastric cardia adenocarcinoma, whereas abdominal obesity was found to be associated with increased EAC risk; even in people with normal BMI.
PMCID: PMC3504700  PMID: 22174193
adenocarcinoma; epidemiology; esophageal cancer; gastric adenocarcinoma; obesity
9.  Dietary fat and meat intakes and risk of reflux esophagitis, Barrett’s esophagus and esophageal adenocarcinoma 
The aim of this study was to investigate whether dietary fat and meat intakes are associated with reflux esophagitis (RE), Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC). In this all-Ireland case-control study, dietary intake data was collected using a food frequency questionnaire in 219 RE patients, 220 BE patients, 224 EAC patients, and 256 frequency-matched controls between 2002 and 2005. Unconditional multiple logistic regression analysis was used to examine the association between dietary variables and disease risk using quartiles of intake, to attain odds ratios (OR) and 95% confidence intervals (95%CI), while adjusting for potential confounders. Patients in the highest quartile of total fat intake had a higher risk of RE (OR=3.54; 95%CI=1.32–9.46) and EAC (OR=5.44; 95%CI=2.08–14.27). A higher risk of RE and EAC was also reported for patients in the highest quartile of saturated fat intake (OR=2.79; 95%CI=1.11–7.04; OR=2.41; 95%CI=1.14–5.08, respectively) and monounsaturated fat intake (OR=2.63; 95%CI=1.01–6.86; OR=5.35; 95%CI=2.14–13.34, respectively). Patients in the highest quartile of fresh red meat intake had a higher risk of EAC (OR=3.15; 95%CI=1.38–7.20). Patients in the highest category of processed meat intake had a higher risk of RE (OR=4.67; 95%CI=1.71–12.74). No consistent associations were seen for BE with either fat or meat intakes. Further studies, investigating the association between dietary fat and food sources of fat are needed to confirm these results.
PMCID: PMC3144995  PMID: 21455992
adenocarcinoma; Barrett’s esophagus; dietary fat; epidemiology; meat
10.  Iron intake and markers of iron status and risk of Barrett’s esophagus and esophageal adenocarcinoma 
Cancer causes & control : CCC  2010;21(12):2269-2279.
To investigate the association between iron intake and iron status with Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC).
220 BE patients, 224 EAC patients, and 256 frequency-matched controls completed a lifestyle and food frequency questionnaire, and provided serum and toenail samples between 2002 and 2005. Using multiple logistic regression, odds ratios (OR) and 95% confidence intervals (95%CI) were calculated within quartiles of intake/status.
Comparing the fourth to the first quartile, ferritin (OR 0.47; 95%CI: 0.23, 0.97) and transferrin saturation (OR 0.41; 95%CI: 0.20, 0.82) were negatively associated with BE; whilst total iron binding capacity was positively associated per 50 µg/dl increment (OR 1.47; 95%CI: 1.12, 1.92). Comparing the fourth to the first quartile, iron intake (OR 0.50; 95%CI: 0.25, 0.98), non-heme iron intake per 10 mg/day increment (OR 0.29; 95%CI: 0.08, 0.99), and toenail iron (OR 0.40; 95%CI: 0.17, 0.93) were negatively associated with EAC; whilst heme iron intake was positively associated (OR 3.11 95%CI: 1.46, 6.61).
Principal conclusion
In contrast to the hypothesis that increased iron intakes and higher iron stores are a risk factor for BE and EAC, this study suggests that higher iron intakes and stores may have a protective association with BE and EAC, with the exception of what was found for heme iron intake.
PMCID: PMC3438890  PMID: 20936528
adenocarcinoma; Barrett's esophagus; epidemiology; ferritin; iron
11.  Risk Stratification of Latent Tuberculosis Defined by Combined Interferon Gamma Release Assays 
PLoS ONE  2012;7(8):e43285.
Most individuals infected with Mycobacterium tuberculosis develop latent tuberculosis infection (LTBI). Some may progress to active disease and would benefit from preventive treatment yet no means currently exists to predict who will reactivate. Here, we provide an approach to stratify LTBI based on IFN-γ responses to two antigens, the recombinant Early-Secreted Antigen Target-6 (rESAT-6) and the latency antigen Heparin-Binding Haemagglutinin (HBHA).
We retrospectively analyzed results from in-house IFN-γ-release assays with HBHA (HBHA-IGRA) and rESAT-6 (rESAT-6-IGRA) performed during a 12-year period on serial blood samples (3 to 9) collected from 23 LTBI subjects in a low-TB incidence country. Both the kinetics of the absolute IFN-γ concentrations secreted in response to each antigen and the dynamics of HBHA/rESAT-6-induced IFN-γ concentrations ratios were examined.
This analysis allowed the identification among the LTBI subjects of three major groups. Group A featured stable HBHA and rESAT-6-IGRA profiles with an HBHA/rESAT-6 ratio persistently higher than 1, and with high HBHA- and usually negative rESAT-6-IGRA responses throughout the study. Group B had changing HBHA/rESAT-6 ratios fluctuating from 0.0001 to 10,000, with both HBHA and rESAT-6 responses varying over time at least once during the follow-up. Group C was characterized by a progressive disappearance of all responses.
By combining the measures of IFN-γ concentrations secreted in response to an early and a latency antigens, LTBI subjects can be stratified into different risk groups. We propose that disappearing responses indicate cure, that persistent responses to HBHA with HBHA/rESAT-6 ratios ≥1 represent stable LTBI subjects, whereas subjects with ratios varying from >1 to <1 should be closely monitored as they may represent the highest-risk group, as illustrated by a case report, and should therefore be prioritized for preventive treatment.
PMCID: PMC3422279  PMID: 22912846
12.  Diagnosis of latent tuberculosis infection in healthy young adults in a country with high tuberculosis burden and BCG vaccination at birth 
BMC Research Notes  2012;5:415.
One third of the world’s population is thought to have latent tuberculosis infection (LTBI) with the potential for subsequent reactivation of disease. To better characterize this important population, studies comparing Tuberculin Skin Test (TST) and the new interferon-γ release assays including QuantiFERON®-TB Gold In-Tube (QFT-GIT) have been conducted in different parts of the world, but most of these have been in countries with a low incidence of tuberculosis (TB). The aim of this study was therefore to evaluate the use of QFT-GIT assay as compared with TST in the diagnosis of LTBI in Ethiopia, a country with a high burden of TB and routine BCG vaccination at birth.
Healthy medical and paramedical male students at the Faculty of Medicine, Addis Ababa University, Ethiopia were enrolled into the study from December 2008 to February 2009. The TST and QFTG-IT assay were performed using standard methods.
The mean age of the study participants was 20.9 years. From a total of 107 study participants, 46.7% (95%CI: 37.0% to 56.6%) had a positive TST result (TST≥10 mm), 43.9% (95%CI: 34.3% to 53.9%) had a positive QFT-GIT assay result and 44.9% (95%CI: 35.2% to 54.8%) had BCG scar. There was strong agreement between TST (TST ≥10mm) and QFT-GIT assay (Kappa = 0.83, p value = 0.000).
The TST and QFT-GIT assay show similar efficacy for the diagnosis of LTBI in healthy young adults residing in Ethiopia, a country with high TB incidence.
PMCID: PMC3478185  PMID: 22870897
Tuberculosis; Latent; BCG; Tuberculin skin test; Interferon-γ release assay; Ethiopia
13.  TB case detection: can we remain passive while the process is active? 
TB remains a major public health problem despite all the efforts that have been made since it was declared a global emergency in 1993. Different strategies have been implemented to curb the spread of the epidemic. Early case detection and treatment is one of the pillars of the TB control program. In 1991, WHO set targets for increasing case detection and treatment success rates to 70% and 85% respectively. Although the target of treatment success rate has been achieved, the case detection rate remains far below target at currently less than 50%. It is high time that control programs move from simple passive to a more systematic active case finding in order to accelerate TB control.
PMCID: PMC3343678  PMID: 22593786
Case detection; Tuberculosis; DOTS
14.  Potential of novel Mycobacterium tuberculosis infection phase-dependent antigens in the diagnosis of TB disease in a high burden setting 
Confirming tuberculosis (TB) disease in suspects in resource limited settings is challenging and calls for the development of more suitable diagnostic tools. Different Mycobacterium tuberculosis (M.tb) infection phase-dependent antigens may be differentially recognized in infected and diseased individuals and therefore useful as diagnostic tools for differentiating between M.tb infection states. In this study, we assessed the diagnostic potential of 118 different M.tb infection phase-dependent antigens in TB patients and household contacts (HHCs) in a high-burden setting.
Antigens were evaluated using the 7-day whole blood culture technique in 23 pulmonary TB patients and in 19 to 21 HHCs (total n = 101), who were recruited from a high-TB incidence community in Cape Town, South Africa. Interferon-gamma (IFN-γ) levels in culture supernatants were determined by ELISA.
Eight classical TB vaccine candidate antigens, 51 DosR regulon encoded antigens, 23 TB reactivation antigens, 5 TB resuscitation promoting factors (rpfs), 6 starvation and 24 other stress response-associated TB antigens were evaluated in the study. The most promising antigens for ascertaining active TB were the rpfs (Rv0867c, Rv2389c, Rv2450c, Rv1009 and Rv1884c), with Areas under the receiver operating characteristics curves (AUCs) between 0.72 and 0.80. A combination of M.tb specific ESAT-6/CFP-10 fusion protein, Rv2624c and Rv0867c accurately predicted 73% of the TB patients and 80% of the non-TB cases after cross validation.
IFN-γ responses to TB rpfs show promise as TB diagnostic candidates and should be evaluated further for discrimination between M.tb infection states.
PMCID: PMC3282638  PMID: 22260319
16.  Modulation of Transcriptional and Inflammatory Responses in Murine Macrophages by the Mycobacterium tuberculosis Mammalian Cell Entry (Mce) 1 Complex 
PLoS ONE  2011;6(10):e26295.
The outcome of many infections depends on the initial interactions between agent and host. Aiming at elucidating the effect of the M. tuberculosis Mce1 protein complex on host transcriptional and immunological responses to infection with M. tuberculosis, RNA from murine macrophages at 15, 30, 60 min, 4 and 10 hrs post-infection with M. tuberculosis H37Rv or Δ-mce1 H37Rv was analyzed by whole-genome microarrays and RT-QPCR. Immunological responses were measured using a 23-plex cytokine assay. Compared to uninfected controls, 524 versus 64 genes were up-regulated by 15 min post H37Rv- and Δ-mce1 H37Rv-infection, respectively. By 15 min post-H37Rv infection, a decline of 17 cytokines combined with up-regulation of Ccl24 (26.5-fold), Clec4a2 (23.2-fold) and Pparγ (10.5-fold) indicated an anti-inflammatory response initiated by IL-13. Down-regulation of Il13ra1 combined with up-regulation of Il12b (30.2-fold), suggested switch to a pro-inflammatory response by 4 hrs post H37Rv-infection. Whereas no significant change in cytokine concentration or transcription was observed during the first hour post Δ-mce1 H37Rv-infection, a significant decline of IL-1b, IL-9, IL-13, Eotaxin and GM-CSF combined with increased transcription of Il12b (25.1-fold) and Inb1 (17.9-fold) by 4 hrs, indicated a pro-inflammatory response. The balance between pro-and anti-inflammatory responses during the early stages of infection may have significant bearing on outcome.
PMCID: PMC3200323  PMID: 22039457
17.  Modulation of Cell Death by M. tuberculosis as a Strategy for Pathogen Survival 
It has been clearly demonstrated that in vitro, virulent M. tuberculosis can favor necrosis over apoptosis in infected macrophages, and this has been suggested as a mechanism for evading the host immune response. We recently reported that an effect consistent with this hypothesis could be observed in cells from the blood of TB patients, and in this paper, we review what is known about evasion strategies employed by M. tuberculosis and in particular consider the possible interaction of the apoptosis-inhibiting effects of M. tuberculosis infection with another factor (IL-4) whose expression is thought to play a role in the failure to control M. tuberculosis infection. It has been noted that IL-4 may exacerbate TNF-α-induced pathology, though the mechanism remains unexplained. Since pathology in TB typically involves inflammatory aggregates around infected cells, where TNF-α plays an important role, we predicted that IL-4 would inhibit the ability of cells to remove M. tuberculosis by apoptosis of infected cells, through the extrinsic pathway, which is activated by TNF-α. Infection of human monocytic cells with mycobacteria in vitro, in the presence of IL-4, appears to promote necrosis over apoptosis in infected cells—a finding consistent with its suggested role as a factor in pathology during M. tuberculosis infection.
PMCID: PMC3022200  PMID: 21253484
18.  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.
PMCID: PMC2897260  PMID: 20463103
19.  Immunodiagnosis of tuberculosis: new questions, new tools conference 2008 
BMC Proceedings  2010;4(Suppl 3):I1.
Human infection with Mycobacterium tuberculosis exists as a spectrum of conditions ranging from asymptomatic infection to active disease. Novel, accurate tuberculosis immunodiagnostics have been introduced over the last decade, but it remains challenging to timely diagnose active disease and to accurately distinguish asymptomatic M. tuberculosis infection from immune memory resulting from a prior infection eradicated by the host response. The conference titled Immunodiagnosis of Tuberculosis: New Questions, New Tools, which was held on September 21-23, 2008 in Virginia Beach, Virginia, United States, brought together basic scientists and clinical experts to discuss recent progress in tuberculosis research and diagnosis. Global analyses of M. tuberculosis biology and the host immune response, with emphasis on systems approaches to the study of host-pathogen interactions, were presented. Moreover, conference participants discussed new tests in the pipeline and reviewed new technologies leading to novel assay formats. The discussion included technologies ranging from simple, inexpensive point-of-care tests to automated molecular platforms for detection of multiple infections based on the “lab on a chip” concept. It was also recognized that the utility of any new diagnostic relies on laboratory capacity, accessibility, costs, and test deployment. The conference included lessons from the field. For example, the application of existing technologies to neglected areas, such as diagnosis in children and HIV+ populations, was discussed.
PMCID: PMC3254898  PMID: 21172041
20.  Separating latent and acute TB 
BMC Proceedings  2010;4(Suppl 3):O8.
PMCID: PMC3254917
21.  Two loci control tuberculin skin test reactivity in an area hyperendemic for tuberculosis 
The Journal of Experimental Medicine  2009;206(12):2583-2591.
Approximately 20% of persons living in areas hyperendemic for tuberculosis (TB) display persistent lack of tuberculin skin test (TST) reactivity and appear to be naturally resistant to infection by Mycobacterium tuberculosis. Among those with a positive response, the intensity of TST reactivity varies greatly. The genetic basis of TST reactivity is not known. We report on a genome-wide linkage search for loci that have an impact on TST reactivity, which is defined either as zero versus nonzero (TST-BINa) or as extent of TST in millimeters (TST–quantitative trait locus [QTL]) in a panel of 128 families, including 350 siblings, from an area of South Africa hyperendemic for TB. We detected a major locus (TST1) on chromosomal region 11p14 (P = 1.4 × 10−5), which controls TST-BINa, with a lack of responsiveness indicating T cell–independent resistance to M. tuberculosis. We also detected a second major locus (TST2) on chromosomal region 5p15 (P < 10−5), which controls TST-QTL or the intensity of T cell–mediated delayed type hypersensitivity (DTH) to tuberculin. Fine mapping of this region identified SLC6A3, encoding the dopamine transporter DAT1, as a promising gene for further studies. Our results pave the way for the understanding of the molecular mechanisms involved in resistance to M. tuberculosis infection in endemic areas (TST1) and for the identification of critical regulators of T cell–dependent DTH to tuberculin (TST2).
PMCID: PMC2806605  PMID: 19901083
22.  Immunological Outcomes of New Tuberculosis Vaccine Trials: WHO Panel Recommendations 
PLoS Medicine  2008;5(7):e145.
Willem Hanekom and colleagues make recommendations on assay harmonization for novel tuberculosis vaccine trials.
PMCID: PMC2443198  PMID: 18597551
23.  Immunity against HIV/AIDS, Malaria, and Tuberculosis during Co-Infections with Neglected Infectious Diseases: Recommendations for the European Union Research Priorities 
Author Summary
Infectious diseases remain a major health and socioeconomic problem in many low-income countries, particularly in sub-Saharan Africa. For many years, the three most devastating diseases, HIV/AIDS, malaria, and tuberculosis (TB) have received most of the world's attention. However, in rural and impoverished urban areas, a number of infectious diseases remain neglected and cause massive suffering. It has been calculated that a group of 13 neglected infectious diseases affects over one billion people, corresponding to a sixth of the world's population. These diseases include infections with different types of worms and parasites, cholera, and sleeping sickness, and can cause significant mortality and severe disabilities in low-income countries. For most of these diseases, vaccines are either not available, poorly effective, or too expensive. Moreover, these neglected diseases often occur in individuals who are also affected by HIV/AIDS, malaria, or TB, making the problem even more serious and indicating that co-infections are the rule rather than the exception in many geographical areas. To address the importance of combating co-infections, scientists from 14 different countries in Africa and Europe met in Addis Ababa, Ethiopia, on September 9–11, 2007. The message coming from these scientists is that the only possibility for winning the fight against infections in low-income countries is by studying, in the most global way possible, the complex interaction between different infections and conditions of malnourishment. The new scientific and technical tools of the post-genomic era can allow us to reach this goal. However, a concomitant effort in improving education and social conditions will be needed to make the scientific findings effective.
PMCID: PMC2427178  PMID: 18575596
24.  Ex Vivo Cytokine mRNA Levels Correlate with Changing Clinical Status of Ethiopian TB Patients and their Contacts Over Time 
PLoS ONE  2008;3(1):e1522.
There is an increasing body of evidence which suggests that IL-4 plays a role in the pathogenesis of TB, but a general consensus on its role remains elusive. We have previously published data from a cohort of Ethiopian TB patients, their contacts, and community controls suggesting that enhanced IL-4 production is associated with infection with M. tuberculosis, rather than overt disease and that long-term protection in infected community controls is associated with co-production of the IL-4 antagonist IL-4d2, alongside elevated IL-4. Here, for the first time, we compare data on expression of IFN-γ, IL-4 and IL-4δ2 over time in TB patients and their household contacts. During the follow-up period, the TB patients completed therapy and ceased to display TB-like symptoms. This correlated with a decrease in the relative amount of IL-4 expressed. Over the same period, the clinical status of some of their contacts also changed, with a number developing TB-like symptoms or clinically apparent TB. IL-4 expression was disproportionately increased in this group. The findings support the hypothesis that elevated IL-4 production is generally associated with infection, but that TB disease is associated with a relatively increased expression of IL-4 compared to IFN-γ and IL-4δ2. However, the data also suggest that there are no clear-cut differences between groups: the immune response over time appears to include changes in the expression of IFN-γ, IL-4 and IL-4δ2, and it is the relative, not absolute levels of cytokine expression that are characteristic of clinical status.
PMCID: PMC2211401  PMID: 18231607
25.  Heparin-Binding-Hemagglutinin-Induced IFN-γ Release as a Diagnostic Tool for Latent Tuberculosis 
PLoS ONE  2007;2(10):e926.
The detection of latent tuberculosis infection (LTBI) is a major component of tuberculosis (TB) control strategies. In addition to the tuberculosis skin test (TST), novel blood tests, based on in vitro release of IFN-γ in response to Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 (IGRAs), are used for TB diagnosis. However, neither IGRAs nor the TST can separate acute TB from LTBI, and there is concern that responses in IGRAs may decline with time after infection. We have therefore evaluated the potential of the novel antigen heparin-binding hemagglutinin (HBHA) for in vitro detection of LTBI.
Methodology and Principal Findings
HBHA was compared to purified protein derivative (PPD) and ESAT-6 in IGRAs on lymphocytes drawn from 205 individuals living in Belgium, a country with low TB prevalence, where BCG vaccination is not routinely used. Among these subjects, 89 had active TB, 65 had LTBI, based on well-standardized TST reactions and 51 were negative controls. HBHA was significantly more sensitive than ESAT-6 and more specific than PPD for the detection of LTBI. PPD-based tests yielded 90.00% sensitivity and 70.00% specificity for the detection of LTBI, whereas the sensitivity and specificity for the ESAT-6-based tests were 40.74% and 90.91%, and those for the HBHA-based tests were 92.06% and 93.88%, respectively. The QuantiFERON-TB Gold In-Tube (QFT-IT) test applied on 20 LTBI subjects yielded 50% sensitivity. The HBHA IGRA was not influenced by prior BCG vaccination, and, in contrast to the QFT-IT test, remote (>2 years) infections were detected as well as recent (<2 years) infections by the HBHA-specific test.
The use of ESAT-6- and CFP-10-based IGRAs may underestimate the incidence of LTBI, whereas the use of HBHA may combine the operational advantages of IGRAs with high sensitivity and specificity for latent infection.
PMCID: PMC1991599  PMID: 17912342

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