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Mycobacterium africanum (MAF) is a common cause of human pulmonary tuberculosis in West Africa. We previously described phenotypic differences between MAF and Mycobacterium tuberculosis (MTB) among 290 patients. In the present analysis, we compared 692 tuberculosis patients infected with the two most common lineages within the (MTB) complex found in the Gambia, namely MAF West African type 2 (39% prevalence) and Euro-American MTB (55% prevalence). We identified additional phenotypic differences between infections with these two organisms. MAF patients were more likely to be older and HIV infected. In addition, they had worse disease on chest X-ray, despite complaining of cough for an equal duration, and were more likely severely malnourished. In this cohort, the prevalence of MAF did not change significantly over a 7-year period.

Background: Currently available tools cannot be used to distinguish between sub-species of the M. tuberculosis complex causing latent tuberculosis (TB) infection. M. africanum causes up to half of TB in West- Africa and its relatively lower progression to disease suggests the presence of a large reservoir of latent infection relative to M. tuberculosis., Methods: We assessed the immunogenicity of the TbD1 region, present in M. africanum and absent from "modern" M. tuberculosis, in an ELISPOT assay using cells from confirmed M. africanum or M. tuberculosis infected TB patients without HIV infection in the Gambia., Results: Antigens from the TbD1 region induced IFNgamma responses in only 35% patients and did not discriminate between patients infected with M. africanum vs. M. tuberculosis, while PPD induced universally high responses., Conclusions: Further studies will need to assess other antigens unique to M. africanum that may induce discriminatory immune responses.

Most people infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) actually maintain a strong immune response and are able to control bacterial growth (deemed latently infected (LTBI)), while approximately 10% progress to disease resulting in almost 2 million deaths per year. Determining the immune 'footprint' at specific stages of infection and disease will allow for better diagnostics, treatments and ultimately development of new vaccine candidates. In this study we performed multi-factorial flow cytometry on fresh blood from 56 TB cases, 46 Tuberculin Skin Test (TST) positive (LTBI) and 39 TST negative household contacts. We found a highly significant increase in granulocytes and decrease in B cells and invariant (Valpha24+Vbeta11+) NKT cells in TB cases compared to TST+ contacts (p<0.0001, p=0.007 and p=0.01 respectively) which were restored to LTBI levels following 6 months of TB treatment. Using support vector analysis, we found a combination of granulocyte and lymphocyte and/or NKT cell proportions allowed almost 90% correct classification into M. tuberculosis infection or disease. This work has important public health benefits in regards to diagnosis and treatment of TB in sub-Saharan Africa and in furthering our understanding of the requirements for protective immunity to TB.

Mycobacterium africanum, first described in Senegal in 1968, causes up to half of the smear-positive pulmonary tuberculosis cases in West Africa, but it has not been found in other geographical areas except among recent West African migrants. The reasons for the geographic restriction of M. africanum are unknown. We used molecular tools to determine the population structure of the Mycobacterium tuberculosis complex in a cohort study of consecutive smear-positive tuberculosis cases in The Gambia. We collected and genotyped 386 clinical isolates using spoligotype analysis and PCRs for large sequence polymorphisms (LSPs) and compared the genotype patterns to the patterns in an international database. The results of spoligotyping and LSP analysis for the study population were also compared to determine the correlation between them. The main lineages within the Mycobacterium tuberculosis complex identified in The Gambia included M. africanum type I (38.4%), characterized by an LSP in region of difference 702 (RD702; West African type 2). Among the M. tuberculosis sensu stricto isolates, lineages characterized by RD182 and by RD174 were the most common. We also detected a gradient in the prevalence of M. africanum that extended from neighboring Guinea-Bissau. The genotypic diversity of the spoligotype patterns was greater among the isolates of M. africanum than among the isolates of M. tuberculosis. We postulate that M. africanum became endemic in West Africa first, before the introduction of different lineages within M. tuberculosis sensu stricto.

Tuberculosis (TB) remains a global health problem. The solution involves development of an effective vaccine, but has been limited by incomplete understanding of what constitutes protective immunity during natural infection with Mycobacterium tuberculosis. In this study, M. tuberculosis-specific responses following an overnight whole-blood assay were assessed by intracellular cytokine staining and luminex, and compared between TB cases and exposed household contacts. TB cases had significantly higher levels of IFN-gamma(+)TNF-alpha(+)IL-2(+)CD4(+)T cells compared with contacts. TB cases also had a significantly higher proportion of cells single-positive for TNF-alpha, but lower proportion of cells producing IL-2 alone and these differences were seen for both CD4(+)and CD8(+) T cells. Cytokine profiles from culture supernatants were significantly biased toward a Th1 phenotype (IFN-gamma and IL-12(p40)) together with a complete abrogation of IL-17 secretion in TB cases. Our data indicate that despite a robust response to TB antigens in active TB disease, changes in the pattern of cytokine production between TB infection and disease clearly contribute to disease progression.

Background: There is considerable variability in the outcome of Mycobacterium tuberculosis infection. We hypothesized that Mycobacterium africanum was less likely than M. tuberculosis to transmit and progress to tuberculosis disease., Methods: In a cohort study of patients with tuberculosis and their household contacts in The Gambia, we categorized 1808 HIV-negative tuberculosis contacts according to exposure to M. tuberculosis or M. africanum. Positive skin test results indicated transmission, and development of tuberculosis during 2 years of follow-up indicated progression to disease., Results: Transmission rates were similar, but rates of progression to disease were significantly lower in contacts exposed to M. africanum than in those exposed to M. tuberculosis (1.0% vs. 2.9%; hazard ratio [HR], 3.1 [95% confidence interval {CI}, 1.1-8.7]). Within M. tuberculosis sensu stricto, contacts exposed to a Beijing family strain were most likely to progress to disease (5.6%; HR relative to M. africanum, 6.7 [95% CI, 2.0-22])., Conclusions: M. africanum and M. tuberculosis transmit equally well to household contacts, but contacts exposed to M. africanum are less likely to progress to tuberculosis disease than those exposed to M. tuberculosis. The variable rate of progression by lineage suggests that tuberculosis variability matters in clinical settings and should be accounted for in studies evaluating tuberculosis vaccines and treatment regimens for latent tuberculosis infection.

Background: The BACTEC MGIT 960 was evaluated and compared with BACTEC 9000 MB and Lowenstein-Jensen medium for recovery rate of mycobacteria, time to detection, and contamination rate., Methodology: 147 sputum samples obtained from patients with suspicion of tuberculosis were processed and inoculated into BACTEC MGIT 960, BACTEC 9000 MB and Lowenstein-Jensen medium using standardized procedures., Results: BACTEC MGIT 960 detected 57.1%; BACTEC 9000 MB detected 57.8%; and LJ medium detected 43.5% specimens with Mycobacterium tuberculosis complex (MTBC). BACTEC MGIT 960 had the shortest mean number of days (10.3) to detection, followed by BACTEC 9000 MB (13.2) and LJ medium (26.1). Sign rank test showed all three methods had significant difference in days to detection (each P<0.0001). About 39% of detection by BACTEC MGIT 960 took place within the first week, compared to 27.0% and 0.0% by BACTEC 9000 MB and LJ medium respectively. The best yield was obtained with BACTEC 9000 MB, but when compared with the BACTEC MGIT 960, it was not statistically significant. Performances were the same when the combination of a liquid plus a LJ medium were measured (P=0.05). Contamination rates were significantly higher in BACTEC MGIT 960 (12%) than in BACTEC 9000 MB (7%) (P=0.041) and LJ (4%) medium (P=0.022). BACTEC 9000 MB and LJ medium have lower contamination rates (P=0.607)., Conclusions: BACTEC MGIT 960 had a shorter time to detection of MTBC than BACTEC 9000 MB and L J medium. Despite a higher contamination rate, its performance did not appear to be inferior.

Background: Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis., Methods and Findings: Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body-positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body-positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R(2) = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population., Conclusion: As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.

Antimycobacterial T cell reactivity at different stages of HIV infection was investigated. Subjects were screened with purified protein derivative (PPD), early secreted antigenic target (ESAT)-6, and culture filtrate protein (CFP)-10 antigens for interferon (IFN)-gamma-producing effector T cell responses by direct ex vivo enzyme-linked immunospot (ELISpot) assay. The proportion of responders to PPD tuberculin decreased with a reduction in CD4 T cell count, whereas the proportion of responder to ESAT-6 and CFP-10 did not. The main sources of IFN-gamma secretion were CD4 cells, and the relative responses to ESAT-6 and CFP-10 significantly increased in HIV-infected patients with decreasing CD4 cell count. This may reflect early signs of reactivation, reinfection, or a restricted, inefficient immune response to Mycobacterium tuberculosis.

Background: IFN-gamma Release Assays (IGRAs) have been licensed for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Their performance may depend on assay format and may vary across populations and settings. We compared the diagnostic performance of an in-house T -cell and commercial whole blood-based IGRAs for the diagnosis of LTBI and TB disease in The Gambia., Methods: Newly diagnosed sputum smear positive cases and their household contacts were recruited. Cases and contacts were bled for IGRA and contacts had a Mantoux skin test. We assessed agreement and discordance between the tests and categorized a contact's level of M. tuberculosis exposure according to where s/he slept relative to a case: the same room, same house or a different house. We assessed the relationship between exposure and test results by multiple logistic regression., Results: In 80 newly diagnosed TB cases, the sensitivity of ELISPOT was 78.7% and for QFT-GIT was 64.0% (p = 0.047). Of 194 household contacts 57.1% and 58.8% were positive for ELISPOT and QFT-GIT respectively. The overall agreement between both IGRAs for LTBI in contacts was 71.4% and there was no significant discordance (p = 0.29). There was significant discordance between the IGRAs and TST. Neither IGRA nor TST had evidence of false positive results because of Bacille Calmette Guérin (BCG) vaccination. However, agreement between QFT-GIT and TST as well as discordance between both IGRAs and TST were associated with BCG vaccination. Both IGRAs responded to the M. tuberculosis exposure gradient and were positively associated with increasing TST induration (p = 0.003 for ELISPOT and p = 0.001 for QFT-GIT)., Conclusion: The ELISPOT test is more sensitive than the QFT-GIT for diagnosing TB disease. The two tests perform similarly in the diagnosis of LTBI in TB contacts. Significant discordance between the two IGRAs and between each and the TST remain largely unexplained.

Background: 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., Conclusions: 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.

Tuberculosis remains a substantial global health problem despite effective drug treatments. The efficacy of BCG, the only available vaccine, is variable, especially in tuberculosis-endemic regions. Recent advances in the development of new vaccines against tuberculosis mean that the first of these are now entering into early clinical trials. A recombinant modified vaccinia virus Ankara expressing a major secreted antigen from Mycobacterium tuberculosis, antigen 85A, was the first new tuberculosis vaccine to enter into clinical trials in September 2002. This vaccine is known as MVA85A. In a series of phase I clinical trials in the UK, MVA85A had an excellent safety profile and was highly immunogenic. MVA85A was subsequently evaluated in a series of phase I trials in The Gambia, a tuberculosis-endemic area in west Africa. This vaccine is the only new subunit tuberculosis vaccine to enter into clinical trials in Africa to date. Here, we discuss some of the issues that were considered in the protocol design of these studies including recruitment, inclusion and exclusion criteria, reimbursement of study participants, and HIV testing. These issues are highly relevant to early clinical trials with all new tuberculosis vaccines in the developing world.

Background: New tools are required to improve tuberculosis (TB) diagnosis and treatment, including enhanced ability to compare new treatment strategies. The ELISPOT assay uses Mycobacterium tuberculosis-specific antigens to produce a precise quantitative readout of the immune response to pathogen. We hypothesized that TB patients in The Gambia would have reduced ELISPOT counts after successful treatment., Methods: We recruited Gambian adults with sputum smear and culture positive tuberculosis for ELISPOT assay and HIV test, and followed them up one year later to repeat testing and document treatment outcome. We used ESAT-6, CFP-10 and Purified Protein Derivative (PPD) as stimulatory antigens. We confirmed the reliability of our assay in 23 volunteers through 2 tests one week apart, comparing within and between subject variation., Results: We performed an ELISPOT test at diagnosis and 12 months later in 89 patients. At recruitment, 70/85 HIV-negative patients (82%) were ESAT-6 or CFP-10 (EC) ELISPOT positive, 77 (90%) were PPD ELISPOT positive. Eighty-two cases (96%) successfully completed treatment: 44 (55%; p < 0.001) were EC ELISPOT negative at 12 months, 17 (21%; p = 0.051) were PPD ELISPOT negative. Sixty (73%) cured cases had a CFP-10 ELISPOT count decrease, 64 (78%) had an ESAT-6 ELISPOT count decrease, 58 (70%) had a PPD ELISPOT count decrease. There was a mean decline of 25, 44 and 47 SFU/2 x 105 cells for CFP-10, ESAT-6 and PPD respectively (p < 0.001 for all). Three of 4 HIV positive patients were cured, all 3 underwent ELISPOT reversion; all 4 not cured subjects (3 HIV-negative, 1 HIV positive) were ESAT-6, CFP-10 and PPD ELISPOT positive at 12 months., Conclusion: Successful tuberculosis treatment is accompanied by a significant reduction in the M. tuberculosis-specific antigen ELISPOT count. The ELISPOT has potential as a proxy measure of TB treatment outcome. Further investigation into the decay kinetics of T-cells with treatment is warranted.

Overlapping peptides of Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 offer increased specificity over the purified protein derivative skin test when they were used in an ex vivo enzyme-linked immunospot (ELISPOT) assay for gamma interferon detection for the diagnosis of M. tuberculosis infection from recent exposure. We assessed whether equivalent results could be obtained for a fusion protein of the two antigens and whether a combined readout would offer increased sensitivity in The Gambia. We studied the ELISPOT assay results for 488 household contacts of 88 sputum smear-positive tuberculosis (TB) cases. The proportions of subjects positive by each test and by the tests combined were assessed across an exposure gradient, defined according to sleeping proximity to a TB case. Eighty-eight (18%) subjects were positive for CFP-10 peptides, 148 (30%) were positive for ESAT-6 peptides, 161 (33%) were positive for both peptides, and 168 (34%) were positive for the fusion protein; 188 (39%) subjects had either a positive result for a peptide or a positive result for the fusion protein. There was reasonable agreement between the peptide and the protein results (kappa statistic = 0.78) and no significant discordance (P = 0.38). There was a strong correlation between the fusion protein and combined peptide spot counts (r = 0.9), and responses to the peptide and the proteins all increased significantly according to M. tuberculosis exposure. The proportion of subjects positive for either the pool of peptides or the fusion protein offered maximum sensitivity, being significantly higher than the proportion of subjects positive for ESAT-6 peptides alone (P = 0.007). A fusion protein of ESAT-6 and CFP-10 is equivalent to overlapping peptides for the diagnosis of latent M. tuberculosis infection. Use of a combination of peptides and fusion protein offers improved sensitivity.

Background: Currently, reliable efficacy markers for assessment of new interventions against tuberculosis (TB) are limited to disease and death. More precise measurement of the human immune response to Mycobacterium tuberculosis infection may be important. A qualitative enzyme-linked immunospot assay (ELISPOT) result for early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) offers improved specificity over the purified protein derivative (PPD) skin test reaction in the detection of M. tuberculosis infection. We evaluated the quantitative ELISPOT and PPD skin test responses to recent M. tuberculosis exposure., Methods: We studied quantitative PPD skin test and PPD ELISPOT results in 1052 healthy household contacts of index patients with cases of sputum smear-positive and culture-positive TB in The Gambia, according to a positive or negative ex vivo interferon gamma ELISPOT response to M. tuberculosis-specific antigens (ESAT-6/CFP-10). We then studied the quantitative PPD skin test and PPD ELISPOT results in patient contacts who had positive ESAT-6/CFP-10 results against a natural exposure gradient according to sleeping proximity to a patient with TB., Results: The number of positive results was significantly greater for both PPD skin test and PPD ELISPOT in ESAT-6/CFP-10-positive subjects, compared with others (P<.0001). However, when quantitative PPD skin test and PPD ELISPOT results were compared in ESAT-6/CFP-10-positive subjects, only the ELISPOT count was sensitive to the exposure gradient, increasing significantly according to exposure (P=.009)., Conclusions: The quantitative ELISPOT response to PPD in specific-antigen-positive contacts of patients with TB reflects the infectious load of M. tuberculosis as a result of recent exposure. This finding offers new possibilities for assessment of the efficacy of new interventions, and adjustment should be made for it when relating the early immune response to progression to disease.

The purified protein derivative (PPD) skin test for Mycobacterium tuberculosis infection lacks specificity. We assessed 2 more specific M. tuberculosis antigens (ESAT-6 and CFP-10) by enzyme-linked immunospot assay (ELISPOT) compared with PPD by ELISPOT and skin test in The Gambia. Of 735 household contacts of 130 sputum smear-positive tuberculosis cases, 476 (65%) tested positive by PPD ELISPOT, 300 (41%) tested positive by PPD skin test, and 218 (30%) tested positive by ESAT-6/CFP-10 ELISPOT. Only 15 (2%) had positive ESAT-6/CFP-10 results and negative PPD results by ELISPOT. With increasing M. tuberculosis exposure, the percentage of subjects who were PPD skin test positive/ESAT-6/CFP-10 ELISPOT negative increased (P<.001), whereas the percentage of subjects who were PPD skin test negative/PPD ELISPOT positive decreased (P=.011). Eighteen (31%) ESAT-6/CFP-10 ELISPOT-positive subjects in the lowest exposure category had negative PPD skin test results. ESAT-6/CFP-10 ELISPOT probably offers increased specificity in the diagnosis of M. tuberculosis infection in this tropical setting of endemicity, at the cost of some sensitivity.

Although most mycobacterial lipids are thought to be associated with the cell envelope, the authors previously observed substantial deposits of intracellular lipophilic material. A Nile-red-based cytological assay was used to determine factors which affect the presence and natural history of intracellular lipophilic inclusions (ILIs) in Mycobacterium smegmatis. Development of ILIs was associated with stationary-phase cultures in broth and with aged (6 days) colonies on agar. Using variants of Youmans' defined medium, the frequency and size of ILIs was observed to be minimal in carbon-poor medium. ILIs were observed to form within 15 min after provision of fatty acids to the medium and after a period of several days in nitrogen-poor medium. Analysis of the non-polar lipid extracts of ILI-rich and -poor preparations indicated that the triacylglycerols (TAGs) were a major component of the inclusions. The acyl substituents of the TAGs varied according to whether they were formed in Middlebrook 7H9 broth, in low-nitrogen Youmans' broth or rapidly after oleic acid supplementation of Youmans' broth. These studies support a storage function for TAGs in mycobacteria in addition to their previously suggested occurrence as components of the cell envelope. To assess a possible role for ILIs in Mycobacterium tuberculosis, a combined acid-fast (Auramine)/Nile red assay was applied to heavily positive sputum samples from patients with tuberculosis. Strong intracellular Nile red signals were obtained from acid-fast cells, indicating that ILI occur in M. tuberculosis in vivo. This may reflect a distinct physiological state of these cells, which it has not been possible to reproduce in vitro. These findings indicate that the uptake of long-chain fatty acids and TAG biosynthetic and degradative pathways are important aspects of mycobacterial lipid metabolism, meriting further investigation.

Dizygotic (DZ) twinning has a genetic component and is common among sub-Saharan Africans; in The Gambia its frequency is up to 3% of live births. Variation in PTX3, encoding Pentraxin 3, a soluble pattern recognition receptor that plays an important role both in innate immunity and in female fertility, has been associated with resistance to Mycobacterium tuberculosis pulmonary disease and to Pseudomonas aeruginosa infection in cystic fibrosis patients. We tested whether PTX3 variants in Gambian women associate with DZ twinning, by genotyping five PTX3 single nucleotide polymorphisms (SNPs) in 130 sister pairs (96 full sibs and 34 half sibs) who had DZ twins. Two, three and five SNP haplotypes differed in frequency between twinning mothers and those without a history of twinning (from P= 0.006 to 3.03e-06 for two SNP and three SNP haplotypes, respectively). Twinning mothers and West African tuberculosis-controls from a previous study shared several frequent haplotypes. Most importantly, our data are consistent with an independently reported association of PTX3 and female fertility in a sample from Ghana. Taken together, these results indicate that selective pressure on PTX3 variants that affect the innate immune response to infectious agents, could also produce the observed high incidence of DZ twinning in Gambians. [ABSTRACT FROM AUTHOR]

Background: M. africanum West African 2 constitutes an ancient lineage of the M. tuberculosis complex that commonly causes human tuberculosis in West Africa and has an attenuated phenotype relative to M. tuberculosis. Methodology/Principal Findings: In search of candidate genes underlying these differences, the genome of M. africanum West African 2 was sequenced using classical capillary sequencing techniques. Our findings reveal a unique sequence, RD900, that was independently lost during the evolution of two important lineages within the complex: the "modern" M. tuberculosis group and the lineage leading to M. bovis. Closely related to M. bovis and other animal strains within the M. tuberculosis complex, M. africanum West African 2 shares an abundance of pseudogenes with M. bovis but also with M. africanum West African clade 1. Comparison with other strains of the M. tuberculosis complex revealed pseudogenes events in all the known lineages pointing toward ongoing genome erosion likely due to increased genetic drift and relaxed selection linked to serial transmission-bottlenecks and an intracellular lifestyle. Conclusions/Significance: The genomic differences identified between M. africanum West African 2 and the other strains of the Mycobacterium tuberculosis complex may explain its attenuated phenotype, and pave the way for targeted experiments to elucidate the phenotypic characteristic of M. africanum. Moreover, availability of the whole genome data allows for verification of conservation of targets used for the next generation of diagnostics and vaccines, in order to ensure similar efficacy in West Africa. Author Summary: Mycobacterium africanum, a close relative of M. tuberculosis, is studied for the following reasons: M. africanum is commonly isolated from West African patients with tuberculosis yet has not spread beyond this region, it is more common in HIV infected patients, and it is less likely to lead to tuberculosis after one is exposed to an infectious case. Understanding this organism's unique biology gets a boost from the decoding of its genome, reported in this issue. For example, genome analysis reveals that M. africanum contains a region shared with "ancient" lineages in the M. tuberculosis complex and other mycobacterial species, which was lost independently from both M. tuberculosis and M. bovis. This region encodes a protein involved in transmembrane transport. Furthermore, M. africanum has lost genes, including a known virulence gene and genes for vitamin synthesis, in addition to an intact copy of a gene that may increase its susceptibility to antibiotics that are insufficiently active against M. tuberculosis. Finally, the genome sequence and analysis reported here will aid in the development of new diagnostics and vaccines against tuberculosis, which need to take into account the differences between M. africanum and other species in order to be effective worldwide. [ABSTRACT FROM AUTHOR]

Background: Vaccination with a recombinant modified vaccinia Ankara expressing antigen 85A from Mycobacterium tuberculosis, MVA85A, induces high levels of cellular immune responses in UK volunteers. We assessed the safety and immunogenicity of this new vaccine in West African volunteers. Methods and Findings: We vaccinated 21 healthy adult male subjects (11 BCG scar negative and 10 BCG scar positive) with MVA85A after screening for evidence of prior exposure to mycobacteria. We monitored them over six months, observing for clinical, haematological and biochemical adverse events, together with assessment of the vaccine induced cellular immune response using ELISPOT and flow cytometry. MVA85A was well tolerated with no significant adverse events. Mild local and systemic adverse events were consistent with previous UK trials. Marked immunogenicity was found whether individuals had a previous BCG scar or not. There was not enhanced immunogenicity in those with a BCG scar, and induced T cell responses were better maintained in apparently BCG-naïve Gambians than previously studied BCG-naïve UK vaccinees. Although responses were predominantly attributable to CD4+ T cells, we also identified antigen specific CD8+ T cell responses, in subjects who were HLA B-35 and in whom enough blood was available for more detailed immunological analysis. Conclusions: These data on the safety and immunogenicity of MVA85A in West Africa support its accelerated development as a promising booster vaccine for tuberculosis. [ABSTRACT FROM AUTHOR]

Summary: Contact investigation is a key component of tuberculosis (TB) control in developed, but not developing, countries. We aimed to measure the prevalence of TB among household contacts of sputum-smear-positive TB cases in The Gambia and to assess the sensitivity of an enzyme-linked immunospot (ELISPOT) assay in this regard. Household contacts of adult smear-positive TB patients were assessed by questionnaire, purified protein derivative (PPD) skin test, ELISPOT assay, physical examination, chest X-ray and sputum/gastric aspirate. Thirty-three TB cases were identified from 2174 of 2381 contacts of 317 adult smear-positive pulmonary TB patients, giving a prevalence of 1518/100000. The cases identified tended to have milder disease than those passively detected. The sensitivity of ESAT-6/CFP-10 ELISPOT test as a screening test for TB disease was estimated as 71%. Fifty-six per cent of contacts with a PPD skin test result ≥10mm induration had detectable responses to ESAT-6/CFP-10 by ELISPOT; 11% with a negative PPD skin test (<10mm) had a positive ESAT-6/CFP-10 response. Active screening for TB among contacts of TB patients may have a role in TB control in The Gambia. These individuals are a high-risk group, and the disease identified is less advanced than that found through passive case detection. An ELISPOT assay was relatively insensitive as a screening test for TB. [Copyright &y& Elsevier]

Background: IFN-γ Release Assays (IGRAs) have been licensed for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Their performance may depend on assay format and may vary across populations and settings. We compared the diagnostic performance of an in-house T -cell and commercial whole blood-based IGRAs for the diagnosis of LTBI and TB disease in The Gambia. Methods: Newly diagnosed sputum smear positive cases and their household contacts were recruited. Cases and contacts were bled for IGRA and contacts had a Mantoux skin test. We assessed agreement and discordance between the tests and categorized a contact's level of M. tuberculosis exposure according to where s/he slept relative to a case: the same room, same house or a different house. We assessed the relationship between exposure and test results by multiple logistic regression. Results: In 80 newly diagnosed TB cases, the sensitivity of ELISPOT was 78.7% and for QFT-GIT was 64.0% (p = 0.047). Of 194 household contacts 57.1% and 58.8% were positive for ELISPOT and QFT-GIT respectively. The overall agreement between both IGRAs for LTBI in contacts was 71.4% and there was no significant discordance (p = 0.29). There was significant discordance between the IGRAs and TST. Neither IGRA nor TST had evidence of false positive results because of Bacille Calmette Gu&erin;rin (BCG) vaccination. However, agreement between QFT-GIT and TST as well as discordance between both IGRAs and TST were associated with BCG vaccination. Both IGRAs responded to the M. tuberculosis exposure gradient and were positively associated with increasing TST induration (p = 0.003 for ELISPOT and p = 0.001 for QFT-GIT). Conclusion: The ELISPOT test is more sensitive than the QFT-GIT for diagnosing TB disease. The two tests perform similarly in the diagnosis of LTBI in TB contacts. Significant discordance between the two IGRAs and between each and the TST remain largely unexplained. [ABSTRACT FROM AUTHOR]

The purified protein derivative (PPD) skin test for Mycobacterium tuberculosis infection lacks specificity. We assessed 2 more specific M. tuberculosis antigens (ESAT-6 and CFP-10) by enzyme-linked immunospot assay (ELISPOT) compared with PPD by ELISPOT and skin test in The Gambia. Of 735 household contacts of 130 sputum smear–positive tuberculosis cases, 476 (65%) tested positive by PPD ELISPOT, 300 (41%) tested positive by PPD skin test, and 218 (30%) tested positive by ESAT-6/CFP-10 ELISPOT. Only 15 (2%) had positive ESAT- 6/CFP-10 results and negative PPD results by ELISPOT. With increasing M. tuberculosis exposure, the percentage of subjects who were PPD skin test positive/ESAT-6/CFP-10 ELISPOT negative increased (P < .001) whereas the percentage of subjects who were PPD skin test negative/PPD ELISPOT positive decreased (Pp ). Eighteen (31%) SAT-6/CFP-10 ELISPOT–positive subjects in the lowest exposure category had negative .011 PPD skin test results. ESAT-6/CFP-10 ELISPOT probably offers increased specificity in the diagnosis of M. tuberculosis infection in this tropical setting of endemicity, at the cost of some sensitivity. [ABSTRACT FROM AUTHOR]

Abstract: Understanding the immunogenicity of BCG in a population where it has failed will facilitate the design of new TB vaccines. We assessed the immunogenicity of M. bovis BCG over 12 months by ELISPOT assay. Forty-one adolescents and young Gambian male adults received a tuberculin skin test (TST) which was followed one week later by BCG vaccination, but the 23 control subjects received neither of these. TST alone significantly induced PPD-specific IFN-γ producing cells. Twenty-three percent of subjects did not respond to BCG, which was associated with higher pre-existing ex vivo response to PPD. Paradoxically, amongst BCG responders there was a correlation between pre-existing response and subsequent response to BCG. We conclude that BCG is immunogenic, but this effector response is short-lived and can be limited in higher pre-existing anti-mycobacterial immunity, suggesting a possible threshold beyond which BCG immunogenicity is inhibited. [Copyright &y& Elsevier]

OBJECTIVE. To compare the enzyme-linked immunospot (ELISPOT) assay with the tuberculin skin test (TST) in children for the diagnosis of Mycobacterium tuberculosis infection in the Gambia. METHODS. We divided child contacts of sputum smear-positive tuberculosis cases into 3 age categories (<5, 5-9, and 10-14 years) and assessed agreement between the 2 tests plus their relationship to prior Bacille Calmette-Guerin (BCG) vaccination. We categorized a child's level of M tuberculosis exposure according to where he/she slept relative to a case: the same room, same house, or a different house. The relationship between exposure and test result was assessed by multiple logistic regression. RESULTS. In child contacts of 287 cases, 225 (32.5%) of 693 were positive by TST and 232 (32.3%) of 718 by ELISPOT. The overall agreement between tests was 83% and the discordance was not significant. Both tests responded to the M tuberculosis exposure gradient in each age category. The percentage of those who were TST positive/ELISPOT negative increased with increasing exposure. At the lowest exposure level, the percentage of ELISPOT-positive children who were TST negative was increased compared with the highest exposure level. Neither test had evidence of false positive results because of BCG. CONCLUSIONS. In Gambian children, the ELISPOT is slightly less sensitive than the TST in the diagnosis of M tuberculosis infection from recent exposure, and neither test is confounded by prior BCG vaccination. Evidence of reduced TST sensitivity in subjects with the lowest known recent M tuberculosis exposure suggests that, when maximal sensitivity is important, the 2 tests may lie best used together. [ABSTRACT FROM AUTHOR]