Davinder P. S. Dosanjh

Dynamic relationship between IFN-gamma and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen load

Distinct IFN-γ and IL-2 profiles of Ag-specific CD4+ T cells have recently been associated with different clinical disease states and Ag loads in viral infections. We assessed the kinetics and functional profile of Mycobacterium tuberculosis Ag-specific T cells secreting IFN-γ and IL-2 in 23 patients with untreated active tuberculosis when bacterial and Ag loads are high and after curative treatment, when Ag load is reduced. The frequencies of M. tuberculosis Ag-specific IFN-γ-secreting T cells declined during 28 mo of follow-up with an average percentage decline of 5.8% per year (p = 0.005), while the frequencies of Ag-specific IL-2-secreting T cells increased during treatment (p = 0.02). These contrasting dynamics for the two cytokines led to a progressive convergence of the frequencies of IFN-γ- and IL-2-secreting cells over 28 mo. Simultaneous measurement of IFN-γ and IL-2 secretion at the single-cell level revealed a codominance of IFN-γ-only secreting and IFN-γ/IL-2 dual secreting CD4+ T cells in active disease that shifted to dominance of IFN-γ/IL-2-secreting CD4+ T cells and newly detectable IL-2-only secreting CD4+ T cells during and after treatment. These distinct T cell functional signatures before and after treatment suggest a novel immunological marker of mycobacterial load and clinical status in tuberculosis that now requires validation in larger prospective studies.

Effect of BCG vaccination on risk of Mycobacterium tuberculosis infection in children with household tuberculosis contact: a prospective community-based study

BACKGROUND Little is known about factors that affect the risk of acquiring infection in children exposed to Mycobacterium tuberculosis. The effect of BCG vaccination has been difficult to ascertain because the tuberculin skin test (TST), until recently the only method for detecting M tuberculosis infection, does not reliably distinguish between tuberculosis infection and BCG vaccination. METHODS We investigated risk factors for tuberculosis infection in 979 child household contacts of 414 adult index patients with sputum smear-positive pulmonary tuberculosis in Istanbul, Turkey. Children were aged up to 16 years (median 7, IQR 3-11) and 770 of 979 (79%) had a BCG scar. A T-cell-based enzyme-linked immunospot assay (ELISpot), which is not confounded by BCG vaccination, and TST were used to assess infection. Independent risk factors for infection were identified through multivariate analysis. FINDINGS Amount of tuberculosis exposure within the household and age (a marker of tuberculosis exposure outside the household) were strongly associated with likelihood of infection as measured by both TST and ELISpot. ELISpot also identified absence of BCG scar as an independent risk factor for infection in tuberculosis-exposed children; BCG-vaccinated children had an odds ratio of 0.60 (95% CI 0.43-0.83, p=0.003) for tuberculosis infection, compared with unvaccinated children. INTERPRETATION Contrary to the prevailing theory that BCG vaccination protects only against tuberculosis disease, our results suggest that the vaccine also protects against tuberculosis infection. This finding has important implications for our understanding of the biology of tuberculosis infection and development of improved tuberculosis vaccines.

Evaluation of T-Cell Responses to Novel RD1- and RD2-Encoded Mycobacterium tuberculosis Gene Products for Specific Detection of Human Tuberculosis Infection

ABSTRACT The tuberculin skin test for diagnosing Mycobacterium tuberculosis infection suffers from antigenic cross-reactivity of purified protein derivative with BCG, resulting in poor specificity in BCG-vaccinated populations. Comparative genomics has identified several genetic regions in M. tuberculosis and M. bovis that are deleted in M. bovis BCG. Proteins encoded in these regions will form the basis of new specific T-cell-based blood tests that do not cross-react with BCG, but only two, early secretory antigen target 6 and culture filtrate protein 10, have been studied in detail in humans. We investigated four novel gene products, encoded by RD2 (Rv1989c) and RD1 (Rv3873, Rv3878, and Rv3879c), that are absent from most or all of the vaccine strains of BCG, respectively. Sixty-seven overlapping peptides were tested in ex vivo gamma interferon enzyme-linked immunospot assays in 49 patients with culture-confirmed tuberculosis and 38 healthy BCG-vaccinated donors. Forty-five percent (95% confidence interval [CI], 31 to 57%) and 53% (95% CI, 39 to 67%) of the tuberculosis patients responded to Rv3879c and Rv3873, respectively, identifying these proteins as major M. tuberculosis T-cell antigens in humans, while 35 and 25% of the patients responded to Rv3878 and Rv1989c, respectively. Of the 38 BCG-vaccinated donors, 1 (2.6%) responded to peptides from Rv3878 and Rv3879c, 3 (7.9%) responded to Rv3873, and none responded to Rv1989c. Exclusion of cross-reactive peptides encoded in conserved motifs of Rv3873, a PPE family member, increased its specificity to 97.4%. The high specificity of Rv3879c peptides and nonconserved Rv3873 sequences, together with their moderate sensitivity in tuberculosis patients, identifies these peptides as candidates for inclusion in new T-cell-based tests for M. tuberculosis infection.

Use of T Cell-Based Diagnosis of Tuberculosis Infection to Optimize Interpretation of Tuberculin Skin Testing for Child Tuberculosis Contacts

BACKGROUND Treatment of recent tuberculosis infection in children aged <2 years is essential, because of high risk of progression to disease, but diagnosis is hindered by the inaccuracy of the tuberculin skin test (TST). More-accurate T cell-based tests of infection could enhance diagnosis by optimizing interpretation of the TST results. METHODS A total of 979 child tuberculosis contacts in Istanbul underwent the TST and enzyme-linked immunospot assay. Using enzyme-linked immunospot test results as a reference standard, we assessed the effect of age and bacille Calmette-Guérin (BCG) vaccination on the sensitivity and specificity of the TST, and we computed the optimal TST cutoff points, using receiver operating characteristic curves. RESULTS With a TST cutoff point of >or=10 mm, the sensitivity of the TST was 66% for children aged <2 years, which was lower than that for older children (P= .006). Specificity was 75% for BCG-vaccinated children, compared with 92% for unvaccinated children (P= .001). Optimal cutoff points improved TST specificity for children with 1 BCG scar, with little loss of sensitivity. Despite the use of optimal cutoff points, TST sensitivity remained <70% for children aged <2 years, specificity remained <87% for BCG-vaccinated children aged >or=2 years, and overall accuracy was low for children with >1 BCG scar. CONCLUSIONS Negative results of the TST cannot exclude tuberculosis infection for child tuberculosis contacts aged <2 years, which supports the use of preventive therapy regardless of the TST results for this age group. In children aged >or=2 years, the accuracy of the TST can be improved by adjustment of cutoff points for BCG-vaccinated children but remains poor for children with >1 BCG scar. This methodology can define optimal TST cutoff points for diagnosis of tuberculosis infection tailored to target populations.

Novel M tuberculosis Antigen-Specific T-Cells Are Early Markers of Infection and Disease Progression

Background Mycobacterium tuberculosis Region-of-Difference-1 gene products present opportunities for specific diagnosis of M. tuberculosis infection, yet immune responses to only two gene-products, Early Secretory Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10), have been comprehensively investigated. Methods T-cell responses to Rv3873, Rv3878 and Rv3879c were quantified by IFN-γ-enzyme-linked-immunospot (ELISpot) in 846 children with recent household tuberculosis exposure and correlated with kinetics of tuberculin skin test (TST) and ESAT-6/CFP-10-ELISpot conversion over six months and clinical outcome over two years. Results Responses to Rv3873, Rv3878, and Rv3879c were present in 20–25% of contacts at enrolment. Rv3873 and Rv3879c responses were associated with and preceded TST conversion (P = 0.02 and P = 0.04 respectively), identifying these antigens as early targets of cell-mediated immunity following M. tuberculosis exposure. Responses to Rv3873 were additionally associated with subsequent ESAT-6/CFP-10-ELISpot conversion (P = 0.04). Responses to Rv3873 and Rv3878 predicted progression to active disease (adjusted incidence rate ratio [95% CI] 3.06 [1.05,8.95; P = 0.04], and 3.32 [1.14,9.71; P = 0.03], respectively). Presence of a BCG-vaccination scar was associated with a 67% (P = 0.03) relative risk reduction for progression to active tuberculosis. Conclusions These RD1-derived antigens are early targets of cellular immunity following tuberculosis exposure and T-cells specific for these antigens predict progression to active tuberculosis suggesting diagnostic and prognostic utility.

Patient Reported Outcomes and Their Importance in the Development of Novel Surgical Techniques

Introduction. Although traditional quality measures such as morbidity and mortality outcomes still pay an important role in the assessment of health care quality, greater emphasis is now being placed on patient-reported outcome measures such as patient satisfaction. This area is especially important for novel surgical technologies such as single-incision laparoscopic surgery (SILS) and natural orifice translumenal endoscopic surgery (NOTES). These new innovations are able to minimize or abolish surgical scarring and are likely to have most benefit in the area of patient satisfaction as opposed to traditional outcome measures. Therefore, it is important to gauge the public opinion regarding these new techniques, as continued public interest can help support further research in this up-and-coming field. Methods. A questionnaire study was carried out with members of the general public. Questions were asked regarding preference for surgical techniques, including open surgery, laparoscopic surgery, NOTES, and SILS, in the situation of acute appendicitis. Results. The questionnaire was completed by 1006 individuals. Results indicated that an established safety profile was necessary before the introduction of these new techniques into general practice. The concept of scarless surgery did appeal to the public, with SILS being the treatment of choice in the scenario of acute appendicitis. Discussion. The patient perspective on health care is an important aspect of health care quality assessment. This is especially important with regard to the development of novel surgical techniques such as SILS and NOTES. With these techniques, the potential benefits are most likely to be found in the realms of reduced scarring and improved patient satisfaction. The findings from this study demonstrate the public’s interest in these new techniques and thus give further support to continued research and development in this area.

BCG and protection against Mycobacterium tuberculosis infection

In their Comment on our Article Christian Lienhardt and Alimuddin Zumla highlight our point that the limitations of the tuberculin skin test have made it difficult to find out whether bacillus Calmette-Guerin (BCG) protects against acquisition of Mycobacterium tuberculosis infection as well as tuberculosis disease. However our use of the ESAT-6/CFP-10 immunodiagnostic test overcame this limitation. Evidence that BCG does not protect against infection in human beings comes from one paper by Sutherland and Lindgren in 1979. This report was a retrospective analysis of a 1961 postmortem study of 83 BCG-vaccinated and 67 BCG-unvaccinated individuals. Macroscopic pathological lesions suggestive of tuberculous foci in the lungs which were used to denote infection were found in a higher proportion of the unvaccinated than vaccinated participants. However the two groups were mismatched for age and therefore potentially also mismatched for tuberculosis exposure. (excerpt)

Increased risk of Mycobacterium tuberculosis infection in household child contacts exposed to passive tobacco smoke.

Risk factors associated with Mycobacterium tuberculosis infection were investigated in a prospective cohort of household child tuberculosis contacts. A significantly increased risk of acquiring infection was associated with exposure to passive cigarette smoke, higher number of index cases, younger age and reduced household monthly income.

BCG and protection against Mycobacterium tuberculosis infection – Authors' reply

We agree with Frank Cobelens and Suzanne Verver that risk of infection among household contacts depends on the duration of infectiousness of the index patient which could differ between the BCG vaccinated and unvaccinated groups owing to healthseeking behaviour. However there was no evidence that the duration of symptoms before treatment was different between the two groups as reported by the index patients (median 2.0 months for both groups p = 0.13) confirming that differences in infection rates between BCG vaccinated and unvaccinated children were not confounded by differences in duration of exposure. (excerpt)