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Background: BCG is used widely as the sole licensed vaccine against tuberculosis, but it has variable efficacy and the reasons for this are still unclear. No reliable biomarkers to predict future protection against, or acquisition of, TB infection following immunisation have been identified. Lessons from BCG could be valuable in the development of effective tuberculosis vaccines. Objectives: Within the Entebbe Mother and Baby Study birth cohort in Uganda, infants received BCG at birth. We investigated factors associated with latent tuberculosis infection (LTBI) and with cytokine response to mycobacterial antigen at age five years. We also investigated whether cytokine responses at one year were associated with LTBI at five years of age. Methods: Blood samples from age one and five years were stimulated using crude culture filtrates of Mycobacterium tuberculosis in a six-day whole blood assay. IFN-γ, IL-5, IL-13 and IL-10 production was measured. LTBI at five years was determined using T-SPOT. TB® assay. Associations with LTBI at five years were assessed using multivariable logistic regression. Multiple linear regression with bootstrapping was used to determine factors associated with cytokine responses at age five years. Results: LTBI prevalence was 9% at age five years. Only urban residence and history of TB contact/disease were positively associated with LTBI. BCG vaccine strain, LTBI, HIV infection, asymptomatic malaria, growth z-scores, childhood anthelminthic treatment and maternal BCG scar were associated with cytokine responses at age five. Cytokine responses at one year were not associated with acquisition of LTBI by five years of age. Conclusion: Although multiple factors influenced anti-myocbacterial immune responses at age five, factors likely to be associated with exposure to infectious cases (history of household contact, and urban residence) dominated the risk of LTBI.
We analysed data from the Entebbe Mother and Baby Study (EMaBS), a randomised double-blinded placebo-controlled trial of anthelminthic treatment in pregnancy and early childhood, conducted in a peri-urban and rural setting by Lake Victoria, Uganda (ISRCTN32849447). EMaBS was established to investigate effects of helminths and their treatment on immune responses to vaccines and on susceptibility to infectious and allergy-related diseases. The design and results of the trial have been reported [25–27]. Briefly, women attending antenatal care at Entebbe hospital were enrolled between April 2003 and November 2005, and randomised to receive single dose albendazole (400 mg) or placebo and praziquantel (40 mg/kg) or placebo in a 2 × 2 factorial design. At age fifteen months, their children were randomised to receive albendazole or placebo quarterly until age five years. In 2008, additional funding was awarded which allowed us to assess children for LTBI at age five. For this analysis, our primary objectives were to investigate (1) factors associated with LTBI at age five years, (2) factors associated with cytokine response to BCG at age five years, and (3) whether cytokine responses at one year of age were associated with acquisition of LTBI by age five years, among children with documented BCG immunisation in infancy. Socio-demographic data, blood and stool samples were obtained at enrolment during pregnancy. Children received routine BCG immunisation (with polio immunisation) at birth. The three BCG vaccine strains used were provided by the National Medical Stores according to availability: BCG-Russia (BCG-I Moscow strain, Serum Institute of India, India); BCG-Bulgaria (BCG-SL 222 Sofia strain, BB-NCIPD Ltd., Bulgaria); and BCG-Danish (BCG-SSI 1331, Statens Seruminstitut, Denmark) [13]. Participants were seen at the clinic for annual visits (at which they gave blood and stool samples and were weighed and measured) and when ill. Maternal history of TB exposure and disease was ascertained during pregnancy. History of TB exposure and disease in the child was ascertained at annual visits and through illness visits. At one and five years of age, cytokine responses to Mycobacterium tuberculosis crude culture filtrate protein (M.tb-cCFP) were assessed. LTBI at age five was assessed using the Interferon gamma release assay (IGRA), T-SPOT.TB® (Oxford Immunotec, Abingdon, UK), for all children in the cohort who turned age five from March 2009 onwards (when the TB sub-study began). Z-scores for weight-for-age, height-for-age and weight-for-height at age five were calculated from World Health Organisation (WHO) growth standards, using WHO Anthro and AnthroPlus macros. The trial was approved by the Science and Ethics Committee of the Uganda Virus Research Institute, Uganda National Council for Science and Technology, and London School of Hygiene and Tropical Medicine. During pregnancy, women gave written, informed consent for their and their child’s participation. The mother, father or guardian gave written informed consent for additional procedures in this study. Cytokine responses were assessed among children who had documented BCG immunisation in infancy and who provided a blood sample at five years of age, using a whole blood assay [12,18]. Briefly, unseparated heparinised blood was diluted to a final concentration of one-in-four (RPMI supplemented with penicillin, streptomycin and glutamine), plated in 96-well plates and stimulated with M.tb-cCFP (10 μg/ml; kindly provided by John Belisle, University of Colorado, Fort Collins, USA), tetanus toxoid (TT) (12 Lf/ml; Statens Seruminstitut, Denmark), phytohaemagglutinin (10 μg/ml; Sigma, UK), or left unstimulated. Supernatants were harvested on day six and frozen at −80 °C until analysed. Supernatant cytokine concentrations were measured by Enzyme Linked Immunosorbent Assay (ELISA) (Becton Dickinson, UK). Cytokine production in unstimulated wells was subtracted from concentrations produced in response to stimulation. Cytokine responses were regarded as positive if greater than the higher of the mean plus two standard deviations of the negative control for all assays and the lowest standard in the assay (IFN-γ > 73 pg/ml; IL-5 > 34 pg/ml; IL-13 > 18 pg/ml; IL-10 > 48 pg/ml at one year [18] and IFN-γ >9 pg/ml; IL-5 > 8 pg/ml; IL-13 > 16 pg/ml; IL-10 > 8 pg/ml at five years). Values below the cut-off were set to zero. Age of infection with cytomegalovirus (CMV) and herpes simplex virus (HSV) were determined by examining for Immunoglobulin G responses by ELISA (DiaSorin, Saluggia, Italy). To avoid confounding of secular trends with assay performance variability, assays were performed in a randomised sequence after completion of sample collection. Stool was examined for helminth ova and Strongyloides larvae using Kato-Katz [28] and charcoal culture [27] methods, respectively. Blood was examined for Mansonella perstans using modified Knott’s method [29] and for malaria by thick blood film and Leishman’s stain. HIV status was determined in mothers and children ≥18 months by rapid antibody test algorithm, and in younger children by polymerase chain reaction [27]. Data were double-entered into Microsoft Access (Redmond, WA, USA) and analysed using Stata v11 (College Station, TX, USA). The sample size was determined for the trial objectives; from the planned enrolment of 2500 women we expected to retain 1046 children in follow-up at age five [25]. Assuming standard deviation of 0.8log10 this sample size would give 80% power to detect a difference in mean cytokine response of 0.14log10 for an exposure with prevalence 50%, at 5% significance level. Outcomes for this analysis were LTBI at age five years determined by T-SPOT.TB® result, and cytokine responses (IFN-γ, IL-5, IL-13 and IL-10) to M.tb-cCFP at age five. Variables considered as exposures were maternal and childhood anthelminthic treatment, maternal socio-demographic characteristics and helminth infections at enrolment; child sex, birth weight, HIV status, illness history, TB exposure/disease, childhood helminth infections, and anthropometry at age five; BCG vaccine strain used for immunisation. In addition, T-SPOT.TB® status was considered as an exposure for age five cytokine responses. Analyses followed a hierarchical causal diagram approach (Fig. 1) [30]: factors at the same level were considered as potential confounders for each other and for proximal factors. Crude associations were examined and a 15% significance level used to decide which factors to consider in multivariable analyses. Anthropometry variables were not included together in multivariable models to avoid collinearity. Unadjusted effects of trial interventions are reported. Conceptual framework. Logistic regression was used to examine associations with LTBI at age five. Cytokine responses were transformed to log10 (concentration + 1) and then analysed using linear regression with bootstrapping to estimate bias corrected accelerated confidence intervals [31]; results were back-transformed to give geometric mean ratios. Spearman’s correlation coefficients between cytokines responses at one and five years were calculated. Logistic regression was used to investigate whether cytokine responses at one year were associated with LTBI at five years, restricting to children with history of TB exposure or disease (which implies exposure) between one and five years.
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