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1.  Tuberculin Skin Test Reversion following Isoniazid Preventive Therapy Reflects Diversity of Immune Response to Primary Mycobacterium tuberculosis Infection 
PLoS ONE  2014;9(5):e96613.
Healthy household contacts (HHC) of individuals with Tuberculosis (TB) with Tuberculin Skin Test (TST) conversions are considered to harbor latent Mycobacterium tuberculosis (Mtb), and at risk for TB. The immunologic, clinical, and public health implications of TST reversions that occur following Isoniazid preventive therapy (IPT) remain controversial.
To measure frequency of TST reversion following IPT, and variation in interferon-gamma (IFN-γ) responses to Mtb, in healthy Ugandan TB HHC with primary Mtb infection evidenced by TST conversion.
Prospective cohort study of healthy, HIV-uninfected, TST-negative TB HHC with TST conversions. Repeat TST was performed 12 months following conversion (3 months following completion of 9 month IPT course) to assess for stable conversion vs. reversion. Whole blood IFN-γ responses to Mtb antigen 85B (MtbA85B) and whole Mtb bacilli (wMtb) were measured in a subset (n = 27 and n = 42, respectively) at enrollment and TST conversion, prior to initiation of IPT.
Of 122 subjects, TST reversion was noted in 25 (20.5%). There were no significant differences in demographic, clinical, or exposure variables between reverters and stable converters. At conversion, reverters had significantly smaller TST compared to stable converters (13.7 mm vs 16.4 mm, respectively; p = 0.003). At enrollment, there were no significant differences in IFN-γ responses to MtbA85B or wMTB between groups. At conversion, stable converters demonstrated significant increases in IFN-γ responses to Ag85B and wMtb compared to enrollment (p = 0.001, p<0.001, respectively), while there were no significant changes among reverters.
TST reversion following IPT is common following primary Mtb infection and associated with unique patterns of Mtb-induced IFN-γ production. We have demonstrated that immune responses to primary Mtb infection are heterogeneous, and submit that prospective longitudinal studies of cell mediated immune responses to Mtb infection be prioritized to identify immune phenotypes protective against development of TB disease.
PMCID: PMC4010490  PMID: 24796677
2.  Broad Adaptive Immune Responses to M. tuberculosis Antigens Precede TST Conversion in Tuberculosis Exposed Household Contacts in a TB-Endemic Setting 
PLoS ONE  2014;9(12):e116268.
The identification of Mycobacterium-tuberculosis (Mtb) infected individuals remains a challenge due to an insufficient understanding of immune responses detected with the current diagnostic tests for latent tuberculosis i.e. the tuberculin skin test (TST) or IFN–γ release assays (IGRAs) and an inability to distinguish infection stages with current immunologic assays. Further classification based on markers other than IFN–γ may help to define markers of early Mtb infection.
We assessed the TST status of Mtb-exposed household contacts at baseline and at 6 months. Contacts were classified into those with initial positive TST (TST+); those with baseline negative TST but TST conversion at 6 months (TST converters, TSTC) and those with persistently negative TST (PTST−). We assessed their short- and long-term immune responses to PPD and ESAT–6/CFP–10 (EC) via IFN–γ ELISPOT and a multiplex cytokine array in relation to TST status and compared them to those of TB cases to identify immune profiles associated with a spectrum of infection stages.
After 1 and 6 days stimulation with EC, 12 cytokines (IFN–γ, IL–2, IP–10, TNF–α, IL–13, IL–17, IL–10, GMCSF, MIP–1β, MCP–3, IL–2RA and IL–1A) were not different in TSTC compared to TST+ suggesting that robust adaptive Mtb-specific immune responses precede TST conversion. Stratifying contacts by baseline IFN–γ ELISPOT to EC in combination with TST results revealed that IP–10 and IL–17 were highest in the group of TST converters with positive baseline ELISPOT, suggesting they might be markers for recent infection.
We describe a detailed analysis of Mtb-specific biomarker profiles in exposed household contacts in a TB endemic area that provides insights into the dynamic immune responses to Mtb infection and may help to identify biomarkers for ‘at-risk’ populations beyond TST and IGRA.
PMCID: PMC4280211  PMID: 25549338
3.  Biomarker Changes Associated with Tuberculin Skin Test (TST) Conversion: A Two-Year Longitudinal Follow-Up Study in Exposed Household Contacts 
PLoS ONE  2009;4(10):e7444.
A high prevalence (50–80%) of Tuberculin Skin Test Positivity (TST+ ≥10 mm indurations) has been reported in TB endemic countries. This pool forms a huge reservoir for new incident TB cases. However, immune biomarkers associated with TST conversion are largely unknown. The objective of this study was to identify immune biomarkers associated with TST conversion after acute Mycobacterium tuberculosis (MTB) exposure.
Methodology/Principal Findings
A 24 month longitudinal study was carried out in a recently MTB exposed cohort of household contacts (HC = 93; 75% TST+). Control group consisted of unexposed community controls (EC = 59; 46%TST+). Cytokine secretion was assessed in whole blood cultures in response to either mycobacterial culture filtrate (CF) antigens or mitogens (PHA or LPS) using Elisa methodology. Compared to the EC group, the HC group at recruitment (Kruskal-Wallis Test) showed significantly suppressed IFN γ (p = 0.0001), raised IL-10 (p = 0.0005) and raised TNF α (p = 0.001) in response to CF irrespective of their TST status. Seventeen TST-HC, showed TST conversion when retested at 6 months. Post TST conversion (paired t tests) significant increases were observed for CF induced IFN γ (p = 0.038), IL-10 (p = 0.001) and IL-6 (p = 0.006). Cytokine responses were also compared in the exposed HC group with either recent infection [(TST converters (N = 17)] or previous infection [TST+ HC (N = 54)] at 0, 6, 12 and 24 months using ANOVA on repeated measures. Significant differences between the exposed HC groups were noted only at 6 months. CF induced IFN γ was higher in previously infected HC group (p = 0.038) while IL-10 was higher in recently infected HC group (p = 0.041). Mitogen induced cytokine secretion showed similar differences for different group.
Our results suggest that TST conversion is associated with early increases in IFN γ and IL-10 responses and precedes latency by several months post exposure.
PMCID: PMC2758599  PMID: 19826490
4.  Clinical and epidemiological characteristics of individuals resistant to M. tuberculosis infection in a longitudinal TB household contact study in Kampala, Uganda 
BMC Infectious Diseases  2014;14:352.
Despite sustained exposure to a person with pulmonary tuberculosis (TB), some M. tuberculosis (Mtb) exposed individuals maintain a negative tuberculin skin test (TST). Our objective was to characterize these persistently negative TST (PTST-) individuals and compare them to TST converters (TSTC) and individuals who are TST positive at study enrollment.
During a TB household contact study in Kampala, Uganda, PTST-, TSTC, and TST + individuals were identified. PTST- individuals maintained a negative TST over a 2 year observation period despite prolonged exposure to an infectious tuberculosis (TB) case. Epidemiological and clinical characteristics were compared, a risk score developed by another group to capture risk for Mtb infection was computed, and an ordinal regression was performed.
When analyzed independently, epidemiological risk factors increased in prevalence from PTST- to TSTC to TST+. An ordinal regression model suggested age (p < 0.01), number of windows (p < 0.01) and people (p = 0.07) in the home, and sleeping in the same room (p < 0.01) were associated with PTST- and TSTC. As these factors do not exist in isolation, we examined a risk score, which reflects an accumulation of risk factors. This compound exposure score did not differ significantly between PTST-, TSTC, and TST+, except for the 5–15 age group (p = 0.009).
Though many individual factors differed across all three groups, an exposure risk score reflecting a collection of risk factors did not differ for PTST-, TSTC and TST + young children and adults. This is the first study to rigorously characterize the epidemiologic risk profile of individuals with persistently negative TSTs despite close exposure to a person with TB. Additional studies are needed to characterize possible epidemiologic and host factors associated with this phenotype.
PMCID: PMC4091673  PMID: 24970328
Transmission risk factors; Latent Mtb infection; Exposure; Household characteristics; PPD test
5.  Whole Blood Interferon-Gamma Responses to Mycobacterium tuberculosis Antigens in Young Household Contacts of Persons with Tuberculosis in Uganda 
PLoS ONE  2008;3(10):e3407.
Due to immunologic immaturity, IFN-γ-producing T cell responses may be decreased in young children compared to adults, thus we hypothesized that IFN-γ responses to mycobacterial antigens in household contacts exposed to Mycobacterium tuberculosis (Mtb) would be impaired in young children relative to adults. The objective of this study was to compare whole blood IFN-γ production in response to mycobacterial antigens between children and adults in Uganda.
Methodology/Principal Findings
We studied household contacts of persons with culture-positive pulmonary tuberculosis (TB) enrolled in a cohort study conducted in Kampala, Uganda. Whole blood IFN-γ production in response to Mtb culture-filtrate antigens was measured by ELISA and compared between infants (<2 years old, n = 80), young children (2 <5 years old, n = 216), older children (5 <15 years old, n = 443) and adults (≥15 years old, n = 528). We evaluated the relationship between IFN-γ responses and the tuberculin skin test (TST), and between IFN-γ responses and epidemiologic factors that reflect exposure to Mtb, and the effect of prior BCG vaccination on IFN-γ responses. Young household contacts demonstrated robust IFN-γ responses comparable to those of adults that were associated with TST and known risk factors for infection. There was no effect of prior BCG immunization on the IFN-γ response.
Young children in a TB endemic setting can mount robust IFN-γ responses generally comparable to those of adults, and as in adults, these responses correlated with the TST and known epidemiologic risk factors for Mtb infection.
PMCID: PMC2560997  PMID: 18923705
6.  Malnutrition and Helminth Infection Affect Performance of an Interferon γ–Release Assay 
Pediatrics  2010;126(6):e1522-e1529.
We sought to compare the tuberculin skin test (TST) to the QuantiFERON-TB Gold In-Tube assay (QFT-IT) and assess the effects of malnourishment and intestinal helminth infection on QFT-IT results.
In this population-based cross-sectional study from Dhaka, Bangladesh, we screened children for latent tuberculosis infection with the QFT-IT and TST. We assess the agreement between the TST and QFT-IT, risk factors associated with indeterminate QFT-IT results, and magnitude of interferon γ (IFN-γ) production.
Three hundred and two children (aged 11–15.3 years) were enrolled, including 93 (30.8%) who were malnourished. Of 251 participants who provided stool samples, 117 (46.6%) were infected with Ascaris lumbricoides and/or Trichuris trichiura. TST results were positive (≥10 mm) for 101 (33.4%) children and negative for 201 (66.6%) children. QFT-IT results were positive for 107 (35.4%) children, negative for 121 (40.1%) children, and indeterminate for 74 (24.5%) children. Agreement between the tests was moderate (κ = 0.55 [95% confidence interval: 0.44–0.65]; P < .0001) when excluding indeterminate results. Children with indeterminate QFT-IT results were separately compared with children with positive and negative QFT-IT results; malnutrition (P = .0006 and .0003), and helminth infection (P = .05 and .02), and the statistical interaction between these 2 terms (P = .03 and .004) were associated with indeterminate results. Higher levels of IFN-γ in response to tuberculosis antigens were associated with positive TST results (P < .0001); lower levels were associated with malnutrition (P = .02).
Malnutrition and helminth infections were associated with indeterminate QFT-IT results. Therefore, the presence of such conditions may limit the interpretability of QFT-IT results in children.
PMCID: PMC3403682  PMID: 21059723
latent tuberculosis infection; interferon γ–release assay; pediatrics; malnutrition; helminth infection
7.  Longitudinal Assessment of an ELISPOT Test for Mycobacterium tuberculosis Infection 
PLoS Medicine  2007;4(6):e192.
Very little longitudinal information is available regarding the performance of T cell-based tests for Mycobacterium tuberculosis infection. To address this deficiency, we conducted a longitudinal assessment of the enzyme-linked immunosorbent spot test (ELISPOT) test in comparison to the standard tuberculin skin test (TST).
Methods and Findings
In tuberculosis (TB) contacts we repeated ELISPOT tests 3 mo (n = 341) and 18 mo (n = 210) after recruitment and TSTs at 18 mo (n = 130). We evaluated factors for association with conversion and reversion and investigated suspected cases of TB. Of 207 ELISPOT-negative contacts, 51 (24.6%) had 3-mo ELISPOT conversion, which was associated with a positive recruitment TST (odds ratio [OR] 2.2, 95% confidence interval [CI] 1.0–5.0, p = 0.048) and negatively associated with bacillus Calmette-Guérin (BCG) vaccination (OR 0.5, 95% CI 0.2–1.0, p = 0.06). Of 134 contacts, 54 (40.2%) underwent 3-mo ELISPOT reversion, which was less likely in those with a positive recruitment TST (OR 0.3, 95% CI 0.1–0.8, p = 0.014). Between 3 and 18 mo, 35/132 (26.5%) contacts underwent ELISPOT conversion and 28/78 (35.9%) underwent ELISPOT reversion. Of the 210 contacts with complete results, 73 (34.8%) were ELISPOT negative at all three time points; 36 (17.1%) were positive at all three time points. Between recruitment and 18 mo, 20 (27%) contacts had ELISPOT conversion; 37 (50%) had TST conversion, which was associated with a positive recruitment ELISPOT (OR 7.2, 95% CI 1.4–37.1, p = 0.019); 18 (32.7%) underwent ELISPOT reversion; and five (8.9%) underwent TST reversion. Results in 13 contacts diagnosed as having TB were mixed, but suggested higher TST sensitivity.
Both ELISPOT conversion and reversion occur after M. tuberculosis exposure. Rapid ELISPOT reversion may reflect M. tuberculosis clearance or transition into dormancy and may contribute to the relatively low reported ELISPOT conversion rate. Therefore, a negative ELISPOT test for M. tuberculosis infection should be interpreted with caution.
Philip Hill and colleagues report that both ELISPOT conversion and reversion occur afterM. tuberculosis exposure in an endemic country and that the ELISPOT results agree poorly with results from the tuberculin skin test.
Editors' Summary
Tuberculosis is a contagious bacterial infection, usually of the lungs. People with active tuberculosis spread the causative bacterium (Mycobacterium tuberculosis) in airborne droplets whenever they cough or sneeze. Most people exposed to M. tuberculosis in this way never become ill—their immune system successfully contains the infection. However, the bacteria remain dormant in the body and can cause disease years later if host immunity declines because of, for example, infection with the human immunodeficiency virus (HIV). Consequently, to control the spread of tuberculosis, individuals who have been in contact with people with active tuberculosis need to be tested for infection with M. tuberculosis and treated with antituberculosis drugs if positive. The standard test for infection is the tuberculin skin test (TST). In this, bacterial antigens (proteins that the immune system recognize as foreign) are injected under the skin. The immune system of infected individuals attacks the antigen and produces a hard swelling at the injection site. Unfortunately, this test does not detect all M. tuberculosis infections and an alternative, laboratory-based test has recently been developed. During M. tuberculosis infections, immune system cells called T lymphocytes produce interferon gamma. This protein activates macrophages, immune system cells that kill bacteria. The ELISPOT (enzyme-linked immunosorbent spot) test measures interferon gamma production by T lymphocytes.
Why Was This Study Done?
Commercial ELISPOT tests are available for the diagnosis of M. tuberculosis infection, but little is known about how they perform when used in repeat tests in individuals or whether the TST or ELISPOT test is better at predicting later development of tuberculosis. In this study, the researchers investigated these questions in a longitudinal assessment of the ELISPOT test in Gambians exposed to active tuberculosis.
What Did the Researchers Do and Find?
The researchers recruited people who had been in contact with active tuberculosis, did ELISPOT tests and TSTs at recruitment, then repeated the ELISPOT test after three months and both tests in some participants after 18 months. They analyzed how often ELISPOT conversion (a change from a negative to a positive result indicating the development of an active immune response) and reversion (a change from a positive to a negative result reflecting clearance of the bacteria or its entry into a dormant state) occurred, whether the TST results mirrored these changes, and which characteristics of the participants were associated with conversion or reversion. A quarter of participants who initially had a negative ELISPOT result had a positive result at three months, a conversion that was associated with a positive TST at recruitment. ELISPOT reversion at three months, by contrast, was associated with an initially negative TST and occurred in nearly half the participants. However, about a third of the participants had negative ELISPOT results at all three time points and a fifth had positive results at all times. Overall, the two tests agreed in 73% and 60% of the participants at recruitment and at 18 months, respectively. Finally, among the 13 contacts who developed active tuberculosis, some were initially positive in both tests but others showed subsequent conversion in one, both or neither test.
What Do These Findings Mean?
These findings indicate that both ELISPOT conversion and reversion occur after initial screening for M. tuberculosis infection. In addition, they suggest that the immune system responses to M. tuberculosis detected by TST and the ELISPOT test occur over different time scales and so the two tests might differ in their ability to detect M. tuberculosis infections at different times after exposure to the bacteria. Because very few contacts developed active tuberculosis, the findings do not indicate which test best predicts disease development after M. tuberculosis infection. Further studies are needed to provide this information and to unravel the complexities of ELISPOT conversion and reversion after exposure to M. tuberculosis. Importantly, however, the high frequency of ELISPOT reversion seen in this study suggests that a negative ELISPOT result may not reflect a lack of infection after exposure to M. tuberculosis and must, therefore, be interpreted with caution.
Additional Information.
Please access these Web sites via the online version of this summary at
The US Centers for Disease Control and Prevention provide fact sheets from the Division of Tuberculosis Elimination about tuberculosis, its testing and diagnosis, and its treatment
MedlinePlus Encyclopedia contains information on tuberculosis and the tuberculin skin test (in English and Spanish)
The American Lung Association offers fact sheets on tuberculosis and on the tuberculin skin test
PMCID: PMC1891317  PMID: 17564487
8.  Performance of a whole blood interferon gamma assay for detecting latent infection with Mycobacterium tuberculosis in children 
Thorax  2006;61(7):616-620.
The diagnosis of latent Mycobacteriumtuberculosis (MTB) infection with a tuberculin skin test (TST) in children is complicated by the potential influence of prior exposure to Bacille Calmette Geurin (BCG) vaccination or environmental mycobacteria. A whole blood assay has recently been developed to quantitatively measure interferon gamma (IFN‐γ) production by lymphocytes specific to the MTB antigens ESAT‐6 and CFP‐10, but its use and assessment in children has been limited. A study was undertaken to compare the performance of the whole blood IFN‐γ assay with the TST in diagnosing latent tuberculosis (TB) infection or TB disease in children in routine clinical practice.
One hundred and six children with a high risk of latent TB infection or TB disease were enrolled in the study. High risk was defined as contact with TB disease, clinical suspicion of TB disease, or recent arrival from an area of high TB prevalence. The whole blood IFN‐γ assay was undertaken in 101 children.
Seventeen (17%) of the 101 assays yielded inconclusive results due to failure of positive or negative control assays. There was poor correlation between the whole blood IFN‐γ assay and the TST (kappa statistic 0.3) with 26 (70%) of the 37 children defined as latent TB infection by TST having a negative whole blood IFN‐γ assay. There were no instances of a positive whole blood IFN‐γ assay with a negative TST. Mitogen (positive) control IFN‐γ responses were significantly correlated with age (Spearman's coefficient = 0.53, p<0.001) and, in children with latent TB infection identified by TST, those with a positive IFN‐γ assay were older (median 12.9 v 6.92 years, respectively, p = 0.007). The whole blood IFN‐γ assay was positive in all nine children with TB disease.
There was poor agreement between the whole blood IFN‐γ assay and TST for the diagnosis of latent TB. The whole blood IFN‐γ assay may have lower sensitivity than the TST in diagnosing TB infection in children. A significant proportion of whole blood IFN‐γ assays fail when used as a screening assay in routine practice.
PMCID: PMC2104654  PMID: 16601088
tuberculosis; interferon gamma; tuberculin skin test; Mantoux; diagnosis; children
9.  Discordance of Tuberculin Skin Test and Interferon Gamma Release Assay in Recently Exposed Household Contacts of Pulmonary TB Cases in Brazil 
PLoS ONE  2014;9(5):e96564.
Interferon-gamma (IFN-γ) release assays (IGRAs) such as the Quantiferon Gold In-tube test are in vitro assays that measure IFN-γ release from T cells in response to M. tuberculosis (Mtb)-specific antigens. Unlike the tuberculin skin test (TST), IGRA is specific and able to distinguish Mtb-infection from BCG vaccination. In this study we evaluated the concordance between TST and IGRA and the efficacy of IGRA in diagnosing new Mtb infection in household contacts (HHC) of pulmonary tuberculosis (PTB) cases. A total of 357 HHC of TB cases in Vitória, Brazil were studied. A TST was performed within 2 weeks following enrollment of the HHC and if negative a second TST was performed at 8-12 weeks. HHC were categorized as initially TST positive (TST+), persistently TST negative (TST-), or TST converters (TSTc), the latter representative of new infection. IGRA was performed at 8–12 weeks following enrollment and the test results were positive in 82% of TST+, 48% of TSTc, and 12% of TST-, indicating poor concordance between the two test results among HHC in each category. Evaluating CXCL10 levels in a subset of IGRA supernatants or lowering the IGRA cutoff value to define a positive test increased agreement between TST and IGRA test results. However, ROC curves demonstrated that this resulted in a trade-off between sensitivity and specificity of IGRA with respect to TST. Together, the findings suggest that until the basis for the discordance between TST and IGRA is fully understood, it may be necessary to utilize both tests to diagnose new Mtb infection in recently exposed HHC. Operationally, in IGRA negative HHC, it may be useful to employ a lower cutoff value for IGRA to allow closer monitoring for potential conversion.
PMCID: PMC4018294  PMID: 24819060
10.  Longitudinal Analysis of QuantiFERON-TB Gold In-Tube in Children with Adult Household Tuberculosis Contact in South Africa: A Prospective Cohort Study 
PLoS ONE  2011;6(10):e26787.
QuantiFERON-TB Gold In Tube (QFT-GIT) is a tool for detecting M. tuberculosis infection. However, interpretation and utility of serial QFT-GIT testing of pediatric tuberculosis (TB) contacts is not well understood. We compared TB prevalence between baseline and 6 months follow-up using QFT-GIT and tuberculin skin testing (TST) in children who were household contacts of adults with pulmonary TB in South Africa, and explored factors associated with QFT-GIT conversions and reversions.
Prospective study with six month longitudinal follow-up.
Among 270 enrolled pediatric contacts, 196 (73%) underwent 6-month follow-up testing. The 6-month prevalence estimate of MTB infection in pediatric contacts increased significantly from a baseline of 29% (79/270, 95%CI [24–35]) to 38% (103/270, 95% CI [32–44], p<0.001) using QFT-GIT; prevalence increased from a baseline of 28% (71/254, 95%CI [23–34]) to 33% (88/263, 95%CI [21–32], p = 0.002) using TST. Prevalence estimates were influenced by thresholds for positivity for TST, but not for QFT-GIT. Among 134 children with a negative or indeterminate baseline QFT-GIT, 24 (18%) converted to positive at follow-up; conversion rates did not differ significantly when using more stringent thresholds to define QFT-GIT conversion. Older age >10 years (AOR 8.9 95%CI [1.1–72]) and baseline TST positivity ≥5 mm (AOR 5.2 95%CI [1.2–23]) were associated with QFT-GIT conversion. Among 62 children with a positive baseline QFT-GIT, 9 (15%) reverted to negative; female gender (AOR 18.5 95%CI [1.1–321]; p = 0.04] was associated with reversion, while children with baseline positive TST were less likely to have QFT-GIT reversion (AOR 0.01 95%CI [0.001–0.24]).
Among pediatric contacts of adult household TB cases in South Africa, prevalence estimates of TB infection increased significantly from baseline to 6 months. Conversions and reversions occurred among pediatric TB contacts using QFT-GIT, but QFT-GIT conversion rates were less influenced by thresholds used for conversions than were TST conversion rates.
PMCID: PMC3204993  PMID: 22066009
11.  Mycobacterium tuberculosis Infection in Young Children: Analyzing the Performance of the Diagnostic Tests 
PLoS ONE  2014;9(5):e97992.
This study evaluated the performance of the Tuberculin Skin Test (TST) and Quantiferon-TB Gold in-Tube (QFT) and the possible association of factors which may modify their results in young children (0–6 years) with recent contact with an index tuberculosis case.
Materials and Methods
A cross-sectional study including 135 children was conducted in Manaus, Amazonas-Brazil. The TST and QFT were performed and the tests results were analyzed in relation to the personal characteristics of the children studied and their relationship with the index case.
The rates of positivity were 34.8% (TST) and 26.7% (QFT), with 14.1% of indeterminations by the QFT. Concordance between tests was fair (Kappa = 0.35 P<0.001). Both the TST and QFT were associated with the intensity of exposure (Linear OR = 1.286, P = 0.005; Linear OR = 1.161, P = 0.035 respectively) with only the TST being associated with the time of exposure (Linear OR = 1.149, P = 0.009). The presence of intestinal helminths in the TST+ group was associated with negative QFT results (OR = 0.064, P = 0.049). In the TST− group lower levels of ferritin were associated with QFT+ results (Linear OR = 0.956, P = 0.036).
Concordance between the TST and QFT was lower than expected. The factors associated with the discordant results were intestinal helminths, ferritin levels and exposure time to the index tuberculosis case. In TST+ group, helminths were associated with negative QFT results suggesting impaired cell-mediated immunity. The TST−&QFT+ group had a shorter exposure time and lower ferritin levels, suggesting that QFT is faster and ferritin may be a potential biomarker of early stages of tuberculosis infection.
PMCID: PMC4039466  PMID: 24879374
12.  Determining Mycobacterium tuberculosis Infection among BCG-Immunised Ugandan Children by T-SPOT.TB and Tuberculin Skin Testing 
PLoS ONE  2012;7(10):e47340.
Children with latent tuberculosis infection (LTBI) represent a huge reservoir for future disease. We wished to determine Mycobacterium tuberculosis (M.tb) infection prevalence among BCG-immunised five-year-old children in Entebbe, Uganda, but there are limited data on the performance of immunoassays for diagnosis of tuberculosis infection in children in endemic settings. We therefore evaluated agreement between a commercial interferon gamma release assay (T-SPOT.TB) and the tuberculin skin test (TST; 2 units RT-23 tuberculin; positive defined as diameter ≥10 mm), along with the reproducibility of T-SPOT.TB on short-term follow-up, in this population.
Methodology/Principal Findings
We recruited 907 children of which 56 were household contacts of TB patients. They were tested with T-SPOT.TB at age five years and then re-examined with T-SPOT.TB (n = 405) and TST (n = 319) approximately three weeks later. The principal outcome measures were T-SPOT.TB and TST positivity. At five years, 88 (9.7%) children tested positive by T-SPOT.TB. More than half of those that were T-SPOT.TB positive at five years were negative at follow-up, whereas 96% of baseline negatives were consistently negative. We observed somewhat better agreement between initial and follow-up T-SPOT.TB results among household TB contacts (κ = 0.77) than among non-contacts (κ = 0.39). Agreement between T-SPOT.TB and TST was weak (κ = 0.28 and κ = 0.40 for T-SPOT.TB at 5 years and follow-up, respectively). Of 28 children who were positive on both T-SPOT.TB tests, 14 (50%) had a negative TST. Analysis of spot counts showed high levels of instability in responses between baseline and follow-up, indicating variability in circulating numbers of T cells specific for certain M.tb antigens.
We found that T-SPOT.TB positives are unstable over a three-week follow-up interval, and that TST compares poorly with T-SPOT.TB, making the categorisation of children as TB-infected or TB-uninfected difficult. Existing tools for the diagnosis of TB infection are unsatisfactory in determining infection among children in this setting.
PMCID: PMC3471887  PMID: 23077594
13.  Innate and Adaptive Immune Responses during Acute M. tuberculosis Infection in Adult Household Contacts in Kampala, Uganda 
Contacts of active pulmonary tuberculosis (TB) patients are at risk for Mycobacterium tuberculosis (MTB) infection. Because most infections are controlled, studies during MTB infection provide insight into protective immunity. We compared immune responses of adult household contacts that did and did not convert the tuberculin skin test (TST). Innate and adaptive immune responses were measured by whole blood assay. Responses of TST converters (TSTC) were compared with persistently TST negative contacts (PTST–) and contacts who were TST+ at baseline (TST+). TLR-2, TLR-4, and IFN-γR responses to IFN-γ did not differ between the groups, nor did γδ T cell responses. T cell responses to MTB antigens differed markedly among TSTC, PTST–, and TST+ contacts. Thus, no differences in innate responses were found among the three household contact groups. However, adaptive T cell responses to MTB antigens did differ before and during MTB infection among PTST–, TSTC, and TST+ contacts.
PMCID: PMC3403758  PMID: 22492155
14.  Ascaris co-infection does not alter malaria-induced anaemia in a cohort of Nigerian preschool children 
Malaria Journal  2013;12:1.
Co-infection with malaria and intestinal parasites such as Ascaris lumbricoides is common. Malaria parasites induce a pro-inflammatory immune response that contributes to the pathogenic sequelae, such as malarial anaemia, that occur in malaria infection. Ascaris is known to create an anti-inflammatory immune environment which could, in theory, counteract the anti-malarial inflammatory immune response, minimizing the severity of malarial anaemia. This study examined whether Ascaris co-infection can minimize the severity of malarial anaemia.
Data from a randomized controlled trial on the effect of antihelminthic treatment in Nigerian preschool-aged (6–59 months) children conducted in 2006–2007 were analysed to examine the effect of malaria and Ascaris co-infection on anaemia severity. Children were enrolled and tested for malaria, helminths and anaemia at baseline, four, and eight months. Six hundred and ninety subjects were analysed in this study. Generalized linear mixed models were used to assess the relationship between infection status and Ascaris and Plasmodium parasite intensity on severity of anaemia, defined as a haemoglobin less than 11 g/dL.
Malaria prevalence ranged from 35-78% over the course of this study. Of the malaria-infected children, 55% were co-infected with Ascaris at baseline, 60% were co-infected four months later and 48% were co-infected eight months later, underlining the persistent prevalence of malaria-nematode co-infections in this population. Over the course of the study the percentage of anaemic subjects in the population ranged between 84% at baseline and 77% at the eight-month time point. The odds of being anaemic were four to five times higher in children infected with malaria compared to those without malaria. Ascaris infection alone did not increase the odds of being anaemic, indicating that malaria was the main cause of anaemia in this population. There was no significant difference in the severity of anaemia between children singly infected with malaria and co-infected with malaria and Ascaris.
In this cohort of Nigerian preschool children, malaria infection was the major contributor to anaemia status. Ascaris co-infection neither exacerbated nor ameliorated the severity of malarial anaemia.
PMCID: PMC3544581  PMID: 23282136
15.  Regional, Household and Individual Factors that Influence Soil Transmitted Helminth Reinfection Dynamics in Preschool Children from Rural Indigenous Panamá 
Few studies have investigated the relative influence of individual susceptibility versus household exposure factors versus regional clustering of infection on soil transmitted helminth (STH) transmission. The present study examined reinfection dynamics and spatial clustering of Ascaris lumbricoides, Trichuris trichiura and hookworm in an extremely impoverished indigenous setting in rural Panamá over a 16 month period that included two treatment and reinfection cycles in preschool children.
Methodology/Principle Findings
Spatial cluster analyses were used to identify high prevalence clusters for each nematode. Multivariate models were then used (1) to identify factors that differentiated households within and outside the cluster, and (2) to examine the relative contribution of regional (presence in a high prevalence cluster), household (household density, asset-based household wealth, household crowding, maternal education) and individual (age, sex, pre-treatment eggs per gram (epg) feces, height-for-age, latrine use) factors on preschool child reinfection epgs for each STH. High prevalence spatial clusters were detected for Trichuris and hookworm but not for Ascaris. These clusters were characterized by low household density and low household wealth indices (HWI). Reinfection epg of both hookworm and Ascaris was positively associated with pre-treatment epg and was higher in stunted children. Additional individual (latrine use) as well as household variables (HWI, maternal education) entered the reinfection models for Ascaris but not for hookworm.
Even within the context of extreme poverty in this remote rural setting, the distinct transmission patterns for hookworm, Trichuris and Ascaris highlight the need for multi-pronged intervention strategies. In addition to poverty reduction, improved sanitation and attention to chronic malnutrition will be key to reducing Ascaris and hookworm transmission.
Author Summary
Control of soil transmitted helminth (STH) infections is of central importance to improving preschool child health because these infections can have long lasting consequences on growth and development. Our study in indigenous Ngäbe preschool children in western Panama was conducted over a period of 16 months. We monitored reinfection dynamics of three STH infections (Ascaris, Trichuris and hookworm) over two reinfection cycles to gain an understanding of regional, household and individual factors that influenced transmission of these infections among preschool children. Despite the rural setting, where virtually all households live under conditions of extreme poverty, we identified spatial clusters of high prevalence of Trichuris and hookworm in the most remote and poorest area, whereas Ascaris was present throughout the study area. Preschool children who were chronically malnourished (low height-for-age) had a higher reinfection burden of Ascaris and hookworm. Household poverty (low relative household wealth and maternal education) and infrequent latrine use were also influential in Ascaris reinfection. This cross-disciplinary analysis of preschool child STH transmission in a poor rural setting provides pertinent information for STH control programs that aim to break the cycle of poverty and infection.
PMCID: PMC3578751  PMID: 23437411
16.  Tuberculin Skin-Test Reactions Are Unaffected by the Severity of Hyperendemic Intestinal Helminth Infections and Co-Infections 
The tuberculin skin test (TST) quantifies cell-mediated immunity to tuberculosis antigens. Helminths suppress cell-mediated immunity, so we studied the effect of helminth infection and deworming on the TST in a randomized, double-blind, placebo-controlled study in an indigenous Amazon community (N = 195). Stool microscopy diagnosed helminths in 98% and co-infection with multiple species in 24% of study subjects. The TST was positive (≥ 10 mm) for 49%, and responses increased with age (P < 0.001), Bacille Calmette Guerin (BCG) vaccination (P = 0.01), and tuberculosis contact (P = 0.05). TST results had no association with helminth-egg concentrations, species, or co-infections (all P > 0.1). One month after deworming with albendazole (three daily 400-mg doses), helminths were reduced, but 63% remained infected with helminths. Albendazole did not cause a change in TST size (P = 0.8) or positivity (P = 0.9) relative to placebo. Thus, TST reactions were unaffected by albendazole therapy that partially cured intestinal helminth infections, and TST interpretation was unaffected by high-burden helminth infections and co-infection with multiple helminth species.
PMCID: PMC2911178  PMID: 20682875
17.  The Tuberculin Skin Test (TST) Is Affected by Recent BCG Vaccination but Not by Exposure to Non-Tuberculosis Mycobacteria (NTM) during Early Life 
PLoS ONE  2010;5(8):e12287.
The tuberculin skin test (TST) is widely used in TB clinics to aid Mycobacterium tuberculosis (M.tb) diagnosis, but the definition and the significance of a positive test in very young children is still unclear. This study compared the TST in Gambian children at 4½ months of age who either received BCG vaccination at birth (Group 1) or were BCG naïve (Group 2) in order to examine the role of BCG vaccination and/or exposure to environmental mycobacteria in TST reactivity at this age. Nearly half of the BCG vaccinated children had a positive TST (≥5 mm) whereas all the BCG naïve children were non-reactive, confirming that recent BCG vaccination affects TST reactivity. The BCG naïve children demonstrated in vitro PPD responses in peripheral blood in the absence of TST reactivity, supporting exposure to and priming by environmental mycobacterial antigens. Group 2 were then vaccinated at 4½ months of age and a repeat TST was performed at 20–28 months of age. Positive reactivity (≥5 mm) was evident in 11.1% and 12.5% infants from Group 1 and Group 2 respectively suggesting that the timing of BCG vaccination had little effect by this age. We further assessed for immune correlates in peripheral blood at 4½ months of age. Mycobacterial specific IFNγ responses were greater in TST responders than in non-responders, although the size of induration did not correlate with IFNγ. However the IFNγ: IL-10 ratio positively correlated with TST induration suggesting that the relationship between PPD induced IFNγ and IL-10 in the peripheral blood may be important in controlling TST reactivity. Collectively these data provide further insights into how the TST is regulated in early life, and how a positive response might be interpreted.
PMCID: PMC2924396  PMID: 20808814
18.  The presence of a booster phenomenon among contacts of active pulmonary tuberculosis cases: a retrospective cohort 
BMC Public Health  2007;7:38.
Assuming a higher risk of latent tuberculosis (TB) infection in the population of Rio de Janeiro, Brazil, in October of 1998 the TB Control Program of Clementino Fraga Filho Hospital (CFFH) routinely started to recommend a two-step tuberculin skin test (TST) in contacts of pulmonary TB cases in order to distinguish a boosting reaction due to a recall of delayed hypersensitivity previously established by infection with Mycobacterium tuberculosis (M.tb) or BCG vaccination from a tuberculin conversion. The aim of this study was to assess the prevalence of boosted tuberculin skin tests among contacts of individuals with active pulmonary tuberculosis (TB).
Retrospective cohort of TB contacts ≥ 12 years old who were evaluated between October 1st, 1998 and October 31st 2001. Contacts with an initial TST ≤ 4 mm were considered negative and had a second TST applied after 7–14 days. Boosting reaction was defined as a second TST ≥ 10 mm with an increase in induration ≥ 6 mm related to the first TST. All contacts with either a positive initial or repeat TST had a chest x-ray to rule out active TB disease, and initially positive contacts were offered isoniazid preventive therapy. Contacts that boosted did not receive treatment for latent TB infection and were followed for 24 months to monitor the development of TB. Statistical analysis of dichotomous variables was performed using Chi-square test. Differences were considered significant at a p < 0.05.
Fifty four percent (572/1060) of contacts had an initial negative TST and 79% of them (455/572) had a second TST. Boosting was identified in 6% (28/455). The mean age of contacts with a boosting reaction was 42.3 ± 21.1 and with no boosting was 28.7 ± 21.7 (p = 0.01). Fifty percent (14/28) of individuals whose test boosted met criteria for TST conversion on the second TST (increase in induration ≥ 10 mm). None of the 28 contacts whose reaction boosted developed TB disease within two years following the TST.
The low number of contacts with boosting and the difficulty in distinguishing boosting from TST conversion in the second TST suggests that the strategy of two-step TST testing among contacts of active TB cases may not be useful. However, this conclusion must be taken with caution because of the small number of subjects followed.
PMCID: PMC1839086  PMID: 17371600
19.  Comparing Interferon-Gamma Release Assays to Tuberculin Skin Test in Thai Children with Tuberculosis Exposure 
PLoS ONE  2014;9(8):e105003.
Data on the performance of interferon-gamma release assays (IGRAs), QuantiFERON TB Gold In-tube (QFNGIT) and T-Spot.TB, in diagnosing tuberculosis (TB) are limited in Southeast Asia. This study aims to compare the performances of the two IGRAs and TST in Thai children with recent TB exposure.
This multicenter, prospective study enrolled children with recent exposure to active TB adults. Children were investigated for active TB. TST was performed and blood collected for T-Spot.TB and QFNGIT.
158 children were enrolled (87% TB-exposed and 13% active TB, mean age 7.2 years). Only 3 children had HIV infection. 66.7% had TST≥10 mm, while 38.6% had TST≥15 mm. 32.5% had positive QFNGIT; 29.9% had positive T-Spot.TB. QFNGIT and T-Spot.TB positivity was higher among children with active TB compared with TB-exposed children. No indeterminate IGRA results were detected. No statistically significant differences between the performances of the IGRAs and TST at the two cut-offs with increasing TB exposure were detected. Concordance for positive IGRAs and TST ranged from 42–46% for TST≥10 mm and 62–67% for TST≥15 mm. On multivariable analyses, exposure to household primary/secondary caregiver with TB was associated with positive QFNGIT. Higher TB contact score and active TB were associated with positive T-Spot.TB.
Both QFNGIT and T-Spot.TB performed well in our Thai pediatric study population. No differences in the performances between tests with increasing TB exposure were found. Due to accessibility and low cost, using TST may more ideal than IGRAs in diagnosing latent and active TB in healthy children in Thailand and other similar settings.
PMCID: PMC4133381  PMID: 25121513
20.  Agreement between QuantiFERON®-TB Gold In-Tube and the tuberculin skin test and predictors of positive test results in Warao Amerindian pediatric tuberculosis contacts 
BMC Infectious Diseases  2014;14:383.
Interferon-gamma release assays have emerged as a more specific alternative to the tuberculin skin test (TST) for detection of tuberculosis (TB) infection, especially in Bacille Calmette-Guérin (BCG) vaccinated people. We determined the prevalence of Mycobacterium tuberculosis infection by TST and QuantiFERON®-TB Gold In-Tube (QFT-GIT) and assessed agreement between the two test methods and factors associated with positivity in either test in Warao Amerindian children in Venezuela. Furthermore, progression to active TB disease was evaluated for up to 12 months.
163 HIV-negative childhood household contacts under 16 years of age were enrolled for TST, QFT-GIT and chest X-ray (CXR). Follow-up was performed at six and 12 months. Factors associated with TST and QFT-GIT positivity were studied using generalized estimation equations logistic regression models.
At baseline, the proportion of TST positive children was similar to the proportion of children with a positive QFT-GIT (47% vs. 42%, p = 0.12). Overall concordance between QFT-GIT and TST was substantial (kappa 0.76, 95% CI 0.46-1.06). Previous BCG vaccination was not associated with significantly increased positivity in either test (OR 0.68, 95% CI 0.32-1.5 for TST and OR 0.51, 95% CI 0.14-1.9 for QFT-GIT). Eleven children were diagnosed with active TB at baseline. QFT-GIT had a higher sensitivity for active TB (88%, 95% CI 47-98%) than TST (55%, 95% CI 24-83%) while specificities were similar (respectively 58% and 55%). Five initially asymptomatic childhood contacts progressed to active TB disease during follow-up.
Replacement of TST by the QFT-GIT for detection of M. tuberculosis infection is not recommended in this resource-constrained setting as test results showed substantial concordance and TST positivity was not affected by previous BCG vaccination. The QFT-GIT had a higher sensitivity than the TST for the detection of TB disease. However, the value of the QFT-GIT as an adjunct in diagnosing TB disease is limited by a high variability in QFT-GIT results over time.
PMCID: PMC4227090  PMID: 25012075
Tuberculosis; Indigenous children; Diagnostics; Child tuberculosis contacts
21.  Tuberculosis skin testing, anergy and protein malnutrition in Peru 
Malnutrition and intestinal parasites cause immunosuppression. This may cause false-negative tuberculin skin tests (TST) and failure to identify tuberculosis (TB) infection.
To assess factors associated with TST positivity and anergy in disadvantaged communities in Peru.
A study of 212 randomly selected adults: 102 in a rural Amazonian village and 110 shanty town residents in urban Lima.
Respectively 52% and 53% of urban and rural jungle populations were TST-positive. Using simultaneous tetanus and candida skin tests, 99% had at least one positive skin test. Generalised anergy was therefore rare, despite frequent intestinal parasitic infection, including 34% helminth infection prevalence in the jungle. TST positivity was associated with age (P = 0.001), known TB contact (P = 0.02) and poor household ventilation (P = 0.007). TST positivity was not significantly associated with crowding, reported past TB, single/multiple BCG vaccination, income, intestinal parasites, dietary factors, body mass index or body fat. Individuals with lower anthropometric body protein, as measured by corrected arm muscle area, were less likely to be TST-positive (P = 0.02), implying that protein malnutrition caused tuberculin-specific anergy.
These results identify the importance of household ventilation for community TB transmission and add to the evidence that protein malnutrition suppresses TB immunity, causing false-negative TST results.
PMCID: PMC2912519  PMID: 16161252
tuberculosis; anergy; protein malnutrition; anthropometry; TST
22.  Recall of intestinal helminthiasis by HIV-infected South Africans and avoidance of possible misinterpretation of egg excretion in worm/HIV co-infection analyses 
Ascariasis and HIV/AIDS are often co-endemic under conditions of poverty in South Africa; and discordant immune responses to the respective infections could theoretically be affecting the epidemic of HIV/AIDS in various ways. It is well-known that sensitisation to helminthic antigens can aggravate or ameliorate several non-helminthic diseases and impair immunisation against cholera, tetanus and tuberculosis. The human genotype can influence immune responses to Ascaris strongly. With these factors in mind, we have started to document the extent of long-term exposure to Ascaris and other helminths in a community where HIV/AIDS is highly prevalent. In more advanced studies, objectives are to analyse relevant immunological variables (e.g. cytokine activity and immunoglobulin levels). We postulate that when Ascaris is hyperendemic, analysis of possible consequences of co-infection by HIV cannot be based primarily on excretion vs non-excretion of eggs.
Recall of worms seen in faeces was documented in relation to the age of adult volunteers who were either seropositive (n = 170) or seronegative (n = 65) for HIV. Reasons for HIV testing, deworming treatments used or not used, date and place of birth, and duration of residence in Cape Town, were recorded. Confidence intervals were calculated both for group percentages and the inter-group differences, and were used to make statistical comparisons.
In both groups, more than 70% of participants were aware of having passed worms, often both when a child and as an adult. Most of the descriptions fitted Ascaris. Evidence for significantly prolonged exposure to helminthic infection in HIV-positives was supported by more recall of deworming treatment in this group (p < 0.05). Over 90% of the participants had moved to the city from rural areas.
There was a long-term history of ascariasis (and probably other helminthic infections) in both of the groups that were studied. In women in the same community, and in children living where housing and sanitation are better, Ascaris sero-prevalence exceeded egg-prevalence by two- and three-fold, respectively. For ongoing and future analyses of possible consequences of co-infection by Ascaris (and/or other helminths) and HIV/AIDS (and/or other bystander conditions), comparisons must be based mainly on disease-related immunological variables. Especially in adults, comparisons cannot be based only on the presence or absence of eggs in excreta.
PMCID: PMC1483828  PMID: 16725057
23.  Tuberculosis contact investigation with a new, specific blood test in a low-incidence population containing a high proportion of BCG-vaccinated persons 
Respiratory Research  2006;7(1):77.
BCG-vaccination can confound tuberculin skin test (TST) reactions in the diagnosis of latent tuberculosis infection.
We compared the TST with a Mycobacterium tuberculosis specific whole blood interferon-gamma assay (QuantiFERON®-TB-Gold In Tube; QFT-G) during ongoing investigations among close contacts of sputum smear positive source cases in Hamburg, Germany.
During a 6-month period, 309 contacts (mean age 28.5 ± 10.5 years) from a total of 15 source cases underwent both TST and QFT-G testing. Of those, 157 (50.8%) had received BCG vaccination and 84 (27.2%) had migrated to Germany from a total of 25 different high prevalence countries (i.e. >20 cases/100,000). For the TST, the positive response rate was 44.3% (137/309), whilst only 31 (10%) showed a positive QFT-G result. The overall agreement between the TST and the QFT-G was low (κ = 0.2, with 95% CI 0.14.-0.23), and positive TST reactions were closely associated with prior BCG vaccination (OR 24.7; 95% CI 11.7–52.5). In contrast, there was good agreement between TST and QFT-G in non-vaccinated persons (κ = 0.58, with 95% CI 0.4–0.68), increasing to 0.68 (95% CI 0.46–0.81), if a 10-mm cut off for the TST was used instead of the standard 5 mm recommended in Germany.
The QFT-G assay was unaffected by BCG vaccination status, unlike the TST. In close contacts who were BCG-vaccinated, the QFT-G assay appeared to be a more specific indicator of latent tuberculosis infection than the TST, and similarly sensitive in unvaccinated contacts. In BCG-vaccinated close contacts, measurement of IFN-gamma responses of lymphocytes stimulated with M. tuberculosis-specific antigen should be recommended as a basis for the decision on whether to perform subsequent chest X-ray examinations or to start treatment for latent tuberculosis infection.
PMCID: PMC1481586  PMID: 16707012
24.  Virologic and immunologic outcome of HAART in Human Immunodeficiency Virus (HIV)-1 infected patients with and without tuberculosis (TB) and latent TB infection (LTBI) in Addis Ababa, Ethiopia 
HIV/TB coinfection remains a major challenge even after the initiation of HAART. Little is known about Mycobacterium tuberculosis (Mtb) specific immune restoration in relation to immunologic and virologic outcomes after long-term HAART during co-infections with latent and active TB.
A total of 232 adults, including 59 HIV patients with clinical TB (HIV + TB+), 125 HIV patients without clinical TB (HIV + TB-), 13 HIV negative active TB patients (HIV-TB+), and 10 HIV negative Tuberculin Skin TST positive (HIV-TST+), and 25 HIV-TST- individuals were recruited. HAART was initiated in 113 HIV + patients (28 TB + and 85 TB-), and anti-TB treatment for all TB cases. CD4+ T-cell count, HIV RNA load, and IFN-γ responses to ESAT-6/CFP-10 were measured at baseline, 6 months (M6), 18 months (M18) and 24 months (M24) after HAART initiation.
The majority of HIV + TB- (70%, 81%, 84%) as well as HIV + TB + patients (60%, 77%, 80%) had virologic success (HIV RNA < 50 copies/ml) by M6, M18 and M24, respectively. HAART also significantly increased CD4+ T-cell counts at 2 years in HIV + TB + (from 110.3 to 289.9 cells/μl), HIV + TB- patients (197.8 to 332.3 cells/μl), HIV + TST- (199 to 347 cells/μl) and HIV + TST + individuals (195 to 319 cells/μl). Overall, there was no significant difference in the percentage of patients that achieved virologic success and in total CD4+ counts increased between HIV patients with and without TB or LTBI. The Mtb specific IFN-γ response at baseline was significantly lower in HIV + TB + (3.6 pg/ml) compared to HIV-TB + patients (34.4 pg/ml) and HIV + TST + (46.3 pg/ml) individuals; and in HIV-TB + patients compared to HIV-TST + individuals (491.2 pg/ml). By M18 on HAART, the IFN-γ response remained impaired in HIV + TB + patients (18.1 pg/ml) while it normalized in HIV + TST + individuals (from 46.3 to 414.2 pg/ml).
Our data show that clinical and latent TB infections do not influence virologic and immunologic outcomes of ART in HIV patients. Despite this, HAART was unable to restore optimal TB responsiveness as measured by Mtb specific IFN-γ response in HIV/TB patients. Improvement of Mtb-specific immune restoration should be the focus of future therapeutic strategies.
PMCID: PMC3718701  PMID: 23842109
HIV; Tuberculosis; HAART
25.  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

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