Optimal Detection of Latent Mycobacterium tuberculosis Infection by Combined Heparin-Binding Hemagglutinin (HBHA) and Early Secreted Antigenic Target 6 (ESAT-6) Whole-Blood Interferon Gamma Release Assays.

Optimal detection of latent tuberculosis (TB) infection (LTBI) remains a challenge, although it is essential to reach the goal of TB elimination. Our objective was to develop and clinically evaluate a user-friendly, 24-h, whole-blood (WB) interferon gamma (IFN-γ) release assay (IGRA) improving the detection of LTBI, compared to available tests. One milliliter of blood was divided into four aliquots and in vitro stimulated for 24 h with two different stage-specific mycobacterial antigens, i.e., heparin-binding hemagglutinin (HBHA) and early secreted antigenic target 6 (ESAT-6), a latency-associated antigen and a bacterial replication-related antigen, respectively, in addition to positive and negative controls. Clinical evaluation was performed on two independent cohorts of carefully selected subjects, i.e., a training cohort of 83 individuals and a validation cohort of 69 individuals. Both cohorts comprised LTBI subjects (asymptomatic people with a positive tuberculin skin test result and potential exposure to TB index cases), patients with active TB (aTB), and noninfected controls. The sensitivity and specificity of the WB-HBHA-IGRA to identify LTBI subjects among asymptomatic individuals were 93%. Combining the results in response to HBHA and ESAT-6 allowed us to identify LTBI subgroups. One group, with IFN-γ responses to HBHA only, was easily differentiated from patients with aTB. The other group, responding to both antigens like the aTB group, is likely at risk to reactivate the infection and should be prioritized for prophylactic anti-TB treatment. The combined WB-IGRA may be offered to clinicians for the selection of LTBI subjects to benefit from prophylactic treatment.

Implementation of latent tuberculosis screening in HIV care centres: evaluation in a low tuberculosis incidence setting.

The screening and treatment of latent tuberculosis infection (LTBI) to prevent active tuberculosis (TB) is recommended by the WHO in all HIV-infected patients. The aim of this study was to evaluate its implementation within Belgium's HIV care. A multiple-choice questionnaire was sent to 55 physicians working in the country's AIDS reference centres. Response rate reached 62%. Only 20% screened all their HIV-infected patients for LTBI. Screening methods used and their interpretation vary from one physician to another. The main barriers to the implementation of LTBI screening and treatment, as perceived by the participants, are lack of sensitivity of screening tools, risks associated with polypharmacy and toxicity of treatment. The poor coverage of LTBI screening reported here and the inconsistency in methods used raises concern. However, this was not unexpected as, in low-TB incidence countries, who, when and how to screen for LTBI remains unclear and published guidelines show important disparities. Recently, a targeted approach in which only HIV-infected patients at highest risk of TB are screened has been suggested. Such a strategy would limit unnecessary exposure to LTBI treatment. This methodology was approved by 80% of the participants and could therefore achieve greater coverage. Its clinical validation is still pending.

Early cellular immune response to a new candidate mycobacterial vaccine antigen in childhood tuberculosis.

The search for novel vaccines against tuberculosis (TB) would benefit from in-depths knowledge of the human immune responses to Mycobacterium tuberculosis (Mtb) infection. Here, we characterised in a low TB incidence country, the immune responses to a new candidate vaccine antigen against TB, the heparin-binding haemagglutinin (HBHA), in young children in contact with an active TB case (aTB). Children with no history of BCG vaccination were compared to those vaccinated at birth to compare the initial immune responses to HBHA with secondary immune responses. Fifty-eight children with aTB and 76 with latent TB infection (LTBI) were included and they were compared to 90 non-infected children. Whereas Mtb-infected children globally secreted more interferon-gamma (IFN-γ) in response to HBHA compared to the non-infected children, these IFN-γ concentrations were higher in previously BCG-vaccinated compared to non-vaccinated children. The IFN-γ concentrations were similar in LTBI and aTB children, but appeared to differ qualitatively. Whereas the IFN-γ secretion induced by native methylated and recombinant non-methylated HBHA were well correlated for aTB, this was not the case for LTBI children. Thus, Mtb-infected young children develop IFN-γ responses to HBHA that are enhanced by prior BCG vaccination, indicating BCG-induced priming, thereby supporting a prime-boost strategy for HBHA-based vaccines. The qualitative differences between aTB and LTBI in their HBHA-induced IFN-γ responses may perhaps be exploited for diagnostic purposes.

Long-incubation-time gamma interferon release assays in response to purified protein derivative, ESAT-6, and/or CFP-10 for the diagnosis of Mycobacterium tuberculosis infection in children.

The diagnosis of childhood active tuberculosis (aTB) and latent Mycobacterium tuberculosis (M. tuberculosis) infection (LTBI) remains a challenge, and the replacement of tuberculin skin tests (TST) with commercialized gamma interferon (IFN-γ) release assays (IGRA) is not currently recommended. Two hundred sixty-six children between 1 month and 15 years of age, 214 of whom were at risk of recent M. tuberculosis infection and 51 who were included as controls, were prospectively enrolled in our study. According to the results of a clinical evaluation, TST, chest X ray, and microbiological assessment, each children was classified as noninfected, having LTBI, or having aTB. Long-incubation-time purified protein derivative (PPD), ESAT-6, and CFP-10 IGRA were performed and evaluated for their accuracy in correctly classifying the children. Whereas both TST and PPD IGRA were suboptimal for detecting aTB, combining the CFP-10 IGRA with a TST or with a PPD IGRA allowed us to detect all the children with aTB with a specificity of 96% for children who were positive for the CFP-10 IGRA. Moreover, the combination of the CFP-10 IGRA and PPD IGRA detected 96% of children who were eventually classified as having LTBI, but a strong IFN-γ response to CFP-10 (defined as >500 pg/ml) was highly suggestive of aTB, at least among the children who were <3 years old. The use of long-incubation-time CFP-10 IGRA and PPD IGRA should help clinicians to quickly identify aTB or LTBI in young children.

Maturation of CD4+ regulatory T lymphocytes and of cytokine secretions in infants born prematurely.

PURPOSE: To characterize the maturation of CD4(+) regulatory T lymphocytes (Treg) and of cytokine productions in preterm infants during their first 16 months of life. METHODS: The proportions of CD4(+) Treg cells, their phenotypic characteristics, and the mitogen-induced cytokine productions by peripheral blood mononuclear cells (PBMC) were analysed in 26 very-preterm infants from 2 to 16 months of age, and compared to results obtained for 17 cord blood mononuclear cells (CBMC) from very-preterm infants, 12 from term infants and to blood samples from 40 adults. RESULTS: High proportion of CD25(+/high)CD4(+) Treg cells was found at birth in preterm CB with a gradual decreased afterwards. However, their percentage at 16 months of age was still higher than in term CB. In contrast to adults, preterm infants were characterized by excellent linear correlations between the proportions of CD25(+/high)CD4(+) and CD25(+/high)FoxP3(+) CD4(+) or CD25(+/high)CD127(low) CD4(+) or CD25(+/high)FoxP3(+)CD127(low) CD4(+)T lymphocytes. CD45RO(+) and HLA-DR(+) expressions were very low on preterm Treg and progressively increased with age. Functionally, preterm compared to term CBMC secreted in response to phytohaemagglutinin lower IFN-γ, higher IL-5 and similar IL-12p70, IL-10, IL-2 and IL-13 concentrations. IFN-γ, IL-12p70 and IL-10 productions were at 16 months still lower than those obtained for adults CONCLUSION: Preterm differed from term CBMC both by their proportion and phenotype of CD4(+) Treg lymphocytes and by their cytokine secretions. Maturation occurred during infancy with similar IFN-γ secretion but with persistently higher proportion of CD4(+) Treg cells in 1 year preterm infants compared to term neonates.

Monocyte-derived interleukin-10 depresses the Bordetella pertussis- specific gamma interferon response in vaccinated infants.

Antigen-specific gamma interferon (IFN-gamma) has been demonstrated to participate in protection against Bordetella pertussis infection. Circulating mononuclear cells from B. pertussis-infected and from pertussis-vaccinated infants secrete high amounts of IFN-gamma after in vitro stimulation by B. pertussis antigens, but with a large variation in the secreted IFN-gamma levels between individuals. We show here that the inhibition of the specific IFN-gamma response can be at least partially attributed to IL-10 secretion by monocytes. This IL-10 secretion was not associated with polymorphisms at positions -1082, -819, and -592 of the IL-10 gene promoter, suggesting that other genetic or environmental factors affect IL-10 expression and secretion.

Cytokine and antibody profiles in 1-year-old children vaccinated with either acellular or whole-cell pertussis vaccine during infancy.

Two different types of pertussis vaccines are currently available to protect children against whooping cough, the first-generation whole-cell (Pw) vaccines and the more recent acellular (Pa) vaccines. Both types provide good protection, yet induce different types of immune responses in 6-month-old infants, with a strong Th1 response induced by Pw vaccines compared to a mixed Th1/Th2 response and a delay in non-specific IFN-gamma secretions after the administration of Pa vaccines. We show here that at 13 months of age, most Pw- or Pa-vaccinated children display Bordetella pertussis-specific T-cell responses, in addition to significant antibody levels, although a higher Th2/Th1 cytokine ratio remained in Pa recipients compared to Pw recipients. In contrast, the proportion of children with tetanus toxin-specific T-cell responses was lower in Pa than in Pw vaccine recipients, although most children had protective anti-tetanus toxin IgG levels. In addition, the global Th2 bias observed in 6-month-old infants vaccinated with a Pa vaccine was normalized at 13 months.