Seroepidemiology of Hepatitis B and C Virus Infections: A Five-Year Retrospective Study among Blood Donors in Saboba District in the Northern Region of Ghana.

METHODS: A five-year hospital-based retrospective study was carried out among 8605 blood donors comprising 8517 males and 88 females using data on blood donors from Saboba Assemblies of God Hospital located in the Saboba District in the Northern Region of Ghana from 2013 to 2017. Blood bank records on HBV and HCV potential blood donors who visited the hospital to donate blood were retrieved. Donor demographic details, i.e., age and gender, were also recovered. Donors who were registered to the hospital but were not residents of the Northern Region were excluded from the study. Donors with incomplete records were also excluded from the study. The data was managed using Microsoft Excel spreadsheet 2016 and analysed using GraphPad Prism statistical software. RESULTS: The overall prevalence of asymptomatic viral hepatitis B and C infections in the general adult population was 9.59% (95% CI: 9.00-10.20) and 12.71% (95% CI: 12.00-13.40), respectively, with an HBV/HCV coinfection rate of 2.23% (95% CI: 1.90-2.60). The number of donors generally declined with advancement in years from 2038 (23.68%) since 2013 to as low as 1169 (13.59%) in 2016, except for 2017 where a sharp increase of 1926 (22.38%) was observed. The first and second highest proportions of donors fell within the age categories of 20-29 (51.53% (4434)) and 30-39 (32.90% (2831)) respectively. The seroprevalence rate of HBV, HCV, and HBV/HCV coinfection rates were generally higher among the female group than those observed among the male category. The year-to-year variation in HBV, HCV, and HBV/HCV infections was statistically significant. The highest year-to-year HBV seropositivity rate was 11.48% in the year 2013, while that for HCV and HBV/HCV coinfection was 16.24% and 5.85%, respectively, both documented in the year 2014. HBV and HBV/HCV coinfection rates were highest among donors aged <20 years old, while HCV seroprevalence was highest among donors aged 50-59 years old. Significantly higher odds of HBV/HCV coinfection (OR = 5.2; 95% CI:3.3-8.1) was observed in the 2014 compared to the year 2013. Donors aged <20years were at higher risks of HBV and HBV/HCV coinfection rates compared to the other age groups. CONCLUSION: The seroprevalence of HBV and HCV among donors in the Saboba District of the Northern Region of Ghana is endemic. The HBV/HCV coinfection rate also raises serious concern owing to its high prevalence rate among the younger age. Intensive public health education coupled with mobile screening and mass vaccination of seronegative individuals is advised so as to help curb further spread of the infection and in effect help safeguard the health status of potential donors in the district.

Immunogenicity of pneumococcal conjugate vaccine formulations containing pneumococcal proteins, and immunogenicity and reactogenicity of co-administered routine vaccines - A phase II, randomised, observer-blind study in Gambian infants.

BACKGROUND: Two conserved pneumococcal proteins, pneumolysin toxoid (dPly) and pneumococcal histidine triad protein D (PhtD), combined with 10 polysaccharide conjugates from the pneumococcal non-typeable Haemophilus influenzae protein D-conjugate vaccine (PHiD-CV) in two investigational pneumococcal vaccine (PHiD-CV/dPly/PhtD) formulations were immunogenic and well-tolerated when administered to Gambian children. Here, we report immunogenicity of the polysaccharide conjugates, and immunogenicity and reactogenicity of co-administered routine vaccines. METHODS: In this phase II, controlled, observer-blind, single-centre study, healthy infants aged 8-10 weeks were randomised (1:1:1:1:1:1) to six groups. Four groups received 3+0 schedule (2-3-4 months [M]) of PHiD-CV/dPly/PhtD (10 or 30 µg of each protein), PHiD-CV, or 13-valent pneumococcal conjugate vaccine; and two groups received 2+1 schedule (2-4-9 M) of PHiD-CV/dPly/PhtD (30 µg of each protein) or PHiD-CV. All infants received diphtheria-tetanus-whole cell pertussis-hepatitis B-Haemophilus influenzae type b (DTPw-HBV/Hib) and oral trivalent polio vaccines (OPV) at 2-3-4 M, and measles, yellow fever, and OPV vaccines at 9 M. We evaluated immune responses at 2-5-9-12 M; and reactogenicity 0-3 days post-vaccination. RESULTS: 1200 infants were enrolled between June 2011 and May 2012; 1152 completed the study. 1 M post-primary vaccination, for each PHiD-CV serotype except 6B and 23F, ≥97.4% (3+0 schedule) and ≥96.4% (2+1 schedule) of infants had antibody concentrations ≥0.2 µg/mL. Immune responses were comparable between groups within the same vaccination schedules. Observed antibody geometric mean concentrations (GMCs) increased by 1 M post-primary vaccination compared to pre-vaccination. In the following months, GMCs and opsonophagocytic activity titres waned, with an increase post-booster for the 2+1 schedule. Immune responses to protein D and, DTPw-HBV/Hib, OPV, measles, and yellow fever vaccines were not altered by co-administration with pneumococcal proteins. Reactogenicity of co-administered vaccines was comparable between groups and did not raise concerns. CONCLUSION: Immune responses to the 10 PHiD-CV polysaccharide conjugates and co-administered vaccines were not altered by addition of dPly and PhtD. ClinicalTrials.gov identifier NCT01262872.

Viral Hepatitis Endemicity and Trends among an Asymptomatic Adult Population in Ho: A 5-Year Retrospective Study at the Ho Municipal Hospital, Ghana.

BACKGROUND: Using prospective blood donors as a proxy, this study was aimed at estimating the burden and five-year (2012-2016) trend of viral hepatitis (HBV and HCV) infection among asymptomatic adult population in Ho. MATERIALS AND METHODS: A retrospective analysis was done on secondary data extracted from the hospital archives comprising 4,180 prospective blood donors from January 2012 to December 2016. Demographic variables included age and sex, as well as place of residence. Screening results of serum infectious markers (HBV and HCV) were obtained. RESULTS: The prevalence of asymptomatic viral hepatitis (HBV and HCV) infection in the general adult population was 6.94% and 1.84%, respectively. Females recorded a higher burden of HBV and HCV (8.3% and 5.0%) compared to their male peers (6.8% and 1.4%). A significant age variation in HBV antigenaemia was seen with HBV seropositivity peaking among the younger population (less than 20 years' group) at 11.24% and troughed among the older population (above 50 years' group) at 0.92%. CONCLUSION: Asymptomatic viral hepatitis among adult population in the Ho Municipality is estimated at the intermediate to high endemicity level. Preventive measures to reduce the burden are urgently needed and should be targeted at the younger generation.

Mycobacterium tuberculosis Infection in Close Childhood Contacts of Adults with Pulmonary Tuberculosis is Increased by Secondhand Exposure to Tobacco.

Tobacco use is a major risk factor for tuberculosis (TB). Secondhand smoke (SHS) is also a risk factor for TB and to a lesser extent, Mycobacterium tuberculosis infection without disease. We investigated the added risk of M. tuberculosis infection due to SHS exposure in childhood contacts of TB cases in The Gambia. Participants were childhood household contacts aged ≤ 14 years of newly diagnosed pulmonary TB (PTB) cases. The intensity of exposure to the case was categorized according to whether contacts slept in the same room, same house, or a different house as the case. Contacts were tested with an enzyme-linked immunospot interferon gamma release assay. In multivariate regression models, M. tuberculosis infection was associated with increasing exposure to a case (odds ratios [OR]: 3.9, 95% confidence interval [CI]: 2.11-71.4, P < 0.001]) and with male gender (OR: 1.5 [95% CI: 1.12-2.11], P = 0.008). Tobacco use caused a 3-fold increase in the odds of M. tuberculosis infection in children who slept closest to a case who smoked within the same home compared with a nonsmoking case (OR: 8.0 [95% CI: 2.74-23.29] versus 2.4 [95% CI: 1.17-4.92], P < 0.001). SHS exposure as an effect modifier appears to greatly increase the risk of M. tuberculosis infection in children exposed to PTB cases. Smoking cessation campaigns may be important for reducing transmission of M. tuberculosis to children within households.

Efficacy of a novel, protein-based pneumococcal vaccine against nasopharyngeal carriage of Streptococcus pneumoniae in infants: A phase 2, randomized, controlled, observer-blind study.

BACKGROUND: Conserved pneumococcal proteins are potential candidates for inclusion in vaccines against pneumococcal diseases. In the first part of a two-part study, an investigational vaccine (PHiD-CV/dPly/PhtD-30) containing 10 pneumococcal serotype-specific polysaccharide conjugates (10VT) combined with pneumolysin toxoid and pneumococcal histidine triad protein D (30µg each) was well tolerated by Gambian children. Part two, presented here, assessed the efficacy of two PHiD-CV/dPly/PhtD formulations against pneumococcal nasopharyngeal carriage (NPC) prevalence in infants. METHODS: In this phase 2, randomized, controlled, observer-blind trial, healthy infants aged 8-10weeks, recruited from a peri-urban health center, were randomized (1:1:1:1:1:1) into six groups. Four groups received PHiD-CV/dPly/PhtD (10 or 30µg of each protein), PHiD-CV, or 13-valent pneumococcal conjugate vaccine at ages 2-3-4months (3+0 infant schedule) and two groups PHiD-CV/dPly/PhtD-30 or PHiD-CV at 2-4-9months (2+1 infant schedule). The primary objective was impact on non-10VT NPC at ages 5-9-12months. Secondary objectives included confirmatory analysis of protein dose superiority and safety/reactogenicity. Impact on pneumococcal NPC acquisition, bacterial load, and ply and phtD gene sequencing were explored. RESULTS: 1200 infants were enrolled between June 2011 and May 2012. Prevalences of pneumococcal (60-67%) and non-10VT (55-61%) NPC were high at baseline. Across all post-vaccination time points, efficacy of PHiD-CV/dPly/PhtD-10 and PHiD-CV/dPly/PhtD-30 against non-10VT NPC (3+0 schedule) was 1.1% (95% CI -21.5, 19.5) and 2.1% (-20.3, 20.3), respectively; efficacy of PHiD-CV/dPly/PhtD-30 (2+1 schedule) was 0.5% (-22.1, 18.9) versus PHiD-CV. No differences were observed in pneumococcal NPC acquisition, clearance, or bacterial load. Both protein-based vaccines elicited immune responses to pneumococcal proteins. CONCLUSIONS: In this high carriage prevalence setting, inclusion of pneumococcal proteins in the PHiD-CV/dPly/PhtD investigational vaccine had no impact on pneumococcal NPC in infants, regardless of protein dose or schedule. Future evaluations will assess its impact against pneumococcal disease endpoints. FUNDING: PATH, GlaxoSmithKline Biologicals SA. ClinicalTrials.gov identifier NCT01262872.

Elevated serum 25-hydroxy (OH) vitamin D levels are associated with risk of TB progression in Gambian adults.

BACKGROUND: Vitamin D is essential in the host defence against tuberculosis (TB) as an immune modulator. The aim of this study was to determine the level of 25-hydroxyvitamin D (25 (OH) D) from adult TB index cases before and after treatment and their exposed household contacts (HHC) in The Gambia. METHODS: Serum from adult index TB cases and their TB-exposed household contacts (HHC) was analysed for 25(OH) D and Vitamin D binding protein (VDBP) concentrations. Tuberculin skin test (TST) status was used as a measure of Mycobacterium tuberculosis (Mtb) infectivity in the HHC. In addition, HHC who later progressed to active TB (incident cases) were assessed alongside non-progressors to determine the influence of 25 (OH) D levels on TB risk. RESULTS: Eighty-three TB cases, 46 TST+ and 52 TST- HHC were analysed. Generally levels of 25(OH) D were considered insufficient in all subjects. However, median levels of 25(OH) D and VDBP were significantly higher in TB cases compared to both TST+ and TST- HHC at recruitment and were significantly reduced after TB therapy (p < 0.0001 for all). In addition, levels of serum 25(OH) D at recruitment were significantly higher in TB progressors compared to non-progressors (median (IQR): 25.0(20.8-29.2) in progressors and 20.3 (16.3-24.6) ng/ml in non-progressors; p = 0.007). CONCLUSION: In The Gambia, an equatorial country, 25(OH) D levels are higher in serum of TB progressors and those with active disease compared to latently infected and uninfected subjects. These results contrast to findings in non-equatorial countries.

Broad adaptive immune responses to M. tuberculosis antigens precede TST conversion in tuberculosis exposed household contacts in a TB-endemic setting.

BACKGROUND: 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. METHODS: 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. RESULTS: 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-1ß, 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. CONCLUSION: 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.

Safety and immunogenicity of the M72/AS01 candidate tuberculosis vaccine when given as a booster to BCG in Gambian infants: an open-label randomized controlled trial.

UNLABELLED: We evaluated the candidate tuberculosis vaccine M72/AS01 in Bacille-Calmette-Guérin (BCG)-vaccinated infants after or concomitantly with Expanded-Programme-on-Immunization (EPI) vaccines. METHODS: In a Phase-II study in The Gambia (NCT01098474), 2 cohorts of 150 BCG-vaccinated infants each were randomized 1:1:1. The 'Outside-EPI' cohort received one or two M72/AS01 doses, or meningitis vaccine, 1-2 months after primary EPI vaccination. The 'Within-EPI' cohort received one or two M72/AS01 doses concomitantly with the third or last two doses of their primary EPI-regimen, respectively, or EPI vaccines alone. Safety, M72-specific humoral (ELISA) and cell-mediated (whole-blood ICS) responses, and humoral responses to EPI vaccines were assessed. RESULTS: M72/AS01 was acceptably tolerated with no vaccine-related serious adverse events reported. Seropositivity/seroprotection rates against EPI antigens in the Within-EPI cohort were comparable between groups, irrespective of M72/AS01 co-administration. Up to one year post M72/AS01 vaccination, M72-specific humoral and CD4(+) T-cell responses were higher after 2 doses versus 1 dose in both cohorts (p < 0.0001), and comparable between cohorts after either 1 or 2 doses (p > 0.05). CONCLUSION: M72/AS01 given to infants after or concomitantly with EPI vaccines had an acceptable safety profile. Our results suggest no interference of immunogenicity profiles occurred following co-administration of M72/AS01 and EPI vaccines. Two M72/AS01 doses elicited higher immune responses than one dose.

Haematological and biochemical reference values of Gambian infants.

OBJECTIVE: To establish haematological and biological reference values for Gambian infants. METHODS: Basic haematological and biochemical indices were analysed in blood samples obtained from healthy infants from Sukuta in the Western Division of The Gambia. The 2.5 and the 97.5 centiles for these indices were estimated. RESULTS: Reference ranges for haematological and biochemical indices were determined. Haemoglobin, total white cell count (WBC) and platelet levels decreased with age (P < 0.001), whereas most of the white cell count subsets except monocytes did not vary with age. Potassium and alkaline phosphatase fell significantly with increasing age (P < 0.001; P < 0.001), whereas urea and creatinine rose with increasing age (P = 0.002; P < 0.001, respectively). CONCLUSION: Our set of haematological and biochemical reference values for healthy infants in The Gambia differs from values in other settings, thus underscoring the importance of establishing region-specific paediatric reference ranges to ensure optimal patient management and evaluate the impact of interventions in clinical research.

Immunogenic Mycobacterium africanum strains associated with ongoing transmission in The Gambia.

In West Africa, Mycobacterium tuberculosis strains co-circulate with M. africanum, and both pathogens cause pulmonary tuberculosis in humans. Given recent findings that M. tuberculosis T-cell epitopes are hyperconserved, we hypothesized that more immunogenic strains have increased capacity to spread within the human host population. We investigated the relationship between the composition of the mycobacterial population in The Gambia, as measured by spoligotype analysis, and the immunogenicity of these strains as measured by purified protein derivative-induced interferon-γ release in ELISPOT assays of peripheral blood mononuclear cells. We found a positive correlation between strains with superior spreading capacity and their relative immunogenicity. Although our observation is true for M. tuberculosis and M. africanum strains, the association was especially pronounced in 1 M. africanum sublineage, characterized by spoligotype shared international type 181, which is responsible for 20% of all tuberculosis cases in the region and therefore poses a major public health threat in The Gambia.

Interferon-gamma treatment kinetics among patients with active pulmonary tuberculosis.

INTRODUCTION: Interferon-γ (IFN-γ) is essential for defence against Mycobacterium tuberculosis; however, levels in patients with active tuberculosis (TB) and changes during treatment have not been documented in our tuberculosis patients in Nigeria, hence this study has been carried out. OBJECTIVE: To determine variations, treatment kinetics, and predictive value of IFN-γ levels during treatment of active tuberculosis. DESIGN: Patients with pulmonary tuberculosis were recruited and subsequently followed up for 3 months during treatment with anti-TB. Peripheral blood was collected for IFN-γ assays, C-reactive protein and others followed by a Mantoux test. IFN-γ levels produced by stimulation with TB antigens were determined by ELISA and repeated measurement of IFN-γ were done at 1 and 3 months of anti-TB therapy. Chi Associations and correlations between IFN-γ were determined. Regression analysis was done to determine association between serial IFN-γ and treatment outcome. RESULTS: We recruited 47 patients with active tuberculosis with a mean age of 34.8 ± 3.6 years and M:F ratio of 1.12:1. Six (11%) were HIV positive. The mean level of IFN-γ induced by TB antigens was 629 ± 114.1 pg/ml, higher for HIV-negative PTB patients compared with HIV-positive PTB patients, 609.78 ± 723.9 pg/ml and 87.88 ± 130.0 pg/ml, respectively, P-value = 0.000. The mean level of IFN-γ induced by TB antigen increased significantly from 629 ± 114.1 pg/ml to 1023.46 + 222.8 pg/ml, P-value = 0.03 and reduced to 272.3 ± 87.7 pg/ml by the third month on anti-TB drugs, P-value = 0.001. Negative correlation was observed between the mean of baseline and chest X-ray involvement, P = 0.03. There was no significant correlation between sputum smear grade with baseline and follow-up IFN-γ levels. Three-month IFN-γ level among cured patients were higher than those with treatment failure, regression analysis showed that it does not predict outcome. CONCLUSION: IFN-γ may be useful in early detection and monitoring response; however, large scale studies are needed.

Interferon-γ ELISPOT as a biomarker of treatment efficacy in latent tuberculosis infection: a clinical trial.

RATIONALE: Biomarkers that can be used to evaluate new interventions against latent tuberculosis infection (LTBI) and predict reactivation TB disease are urgently required. OBJECTIVES: To evaluate ESAT-6 and CFP-10 (EC) IFN-γ ELISPOT as a biomarker for treatment efficacy in LTBI. METHODS: This was a randomized, blinded, and placebo-controlled trial of INH in EC ELISPOT and Mantoux test positive participants. MEASUREMENTS AND MAIN RESULTS: Participants received a 6-month course of 900 mg INH twice weekly or a matching placebo. INH acetylator genotypes were determined and urine tested for INH metabolites to confirm adherence. The proportion of positive responders for CFP-10 and ESAT-6 between treatment arms was compared using mixed effects logistic regression models. A Tweedie (compound Poisson) model was fitted to allow for zero inflation and overdispersion of quantitative response. The proportions of EC ELISPOT-positive subjects reduced over time (P < 0.001) but did not differ by study arm (P = 0.36). Median spot-forming units for ESAT-6 and CFP-10 also declined significantly with time (P < 0.001) but did not differ by study arm (P = 0.74 and 0.71, respectively). There was no evidence of an interaction between acetylator status and INH treatment with respect to ELISPOT results over time. CONCLUSIONS: In contacts with LTBI, INH therapy plays no role in observed decreases in Mycobacterium tuberculosis antigen-specific T-cell responses over time. IFN-γ ELISPOT is probably not a useful biomarker of treatment efficacy in LTBI. Clinical trial registered with www.clinicaltrials.gov (NCT 00130325).

Immunogenicity of the tuberculosis vaccine MVA85A is reduced by coadministration with EPI vaccines in a randomized controlled trial in Gambian infants.

New tuberculosis vaccines are urgently needed to curtail the current epidemic. MVA85A is a subunit vaccine that could enhance immunity from BCG vaccination. To determine MVA85A safety and immunogenicity as well as interactions with other routine vaccines administered in infancy, we randomized healthy 4-month-old infants who had received Bacille Calmette-Guérin at birth to receive Expanded Program on Immunization (EPI) vaccines alone, EPI and MVA85A simultaneously, or MVA85A alone. Adverse events were monitored throughout. Blood samples obtained before vaccination and at 1, 4, and 20 weeks after vaccination were used to assess safety and immunogenicity. The safety profile of both low and standard doses was comparable, but the standard dose was more immunogenic and therefore was selected for the second stage of the study. In total, 72 (first stage) and 142 (second stage) infants were enrolled. MVA85A was safe and well tolerated and induced a potent cellular immune response. Coadministration of MVA85A with EPI vaccines was associated with a significant reduction in MVA85A immunogenicity, but did not affect humoral responses to the EPI vaccines. These results provide important information regarding timing of immunizations, which is required for the design of infant efficacy trials with MVA85A, and suggest that modifications to the standard EPI schedule may be required to incorporate a new generation of T cell-inducing vaccines.

Safety and immunogenicity of the candidate tuberculosis vaccine MVA85A in West Africa.

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. TRIAL REGISTRATION: ClinicalTrials.gov NCT00423839.

The effect of tuberculin skin test and BCG vaccination on the expansion of PPD-specific IFN-gamma producing cells ex vivo.

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-gamma 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.

ESAT-6 and CFP-10 can be combined to reduce the cost of testing for Mycobacterium tuberculosis infection, but CFP-10 responses associate with active disease.

Commercial tests measuring IFN-gamma responses to ESAT-6 and CFP-10 are available for diagnosing Mycobacterium tuberculosis infection. Measures that minimize cost and complexity will facilitate their application in less-developed countries. We investigated whether overlapping peptides representing both ESAT-6 and CFP-10 are required to detect M. tuberculosis infection in a high TB-burden country, and whether they can be combined in a single pool. ESAT-6 and CFP-10 peptides were compared in IFN-gamma enzyme-linked immunospot (ELISPOT) in 183 HIV-negative smear-positive TB cases and 1673 HIV-negative household contacts. Separate peptide pools for each antigen were compared with a combined pool in 498 contacts. Forty per cent of responsive contacts recognized both antigens, 51% only ESAT-6 and 10% only CFP-10, whereas 56% of responsive cases recognized both antigens, 30% only ESAT-6 and 13% only CFP-10. Accordingly, CFP-10 response rates were higher for TB cases (odds ratio 2.409, P<0.001). Low purified protein derivative response rates indicated that responses to CFP-10 only were non-specific in contacts. Agreement between peptides in separate versus combined pools was good (kappa=0.758, r=0.840). Therefore a combined ESAT-6/CFP-10 peptide pool provided maximum sensitivity and efficiency, but CFP-10 was mainly required to detect active disease.

Early clinical trials with a new tuberculosis vaccine, MVA85A, in tuberculosis-endemic countries: issues in study design.

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.

Recognition of stage-specific mycobacterial antigens differentiates between acute and latent infections with Mycobacterium tuberculosis.

Mycobacterium tuberculosis is estimated to infect 80 to 100 million people annually, the majority of whom do not develop clinical tuberculosis (TB) but instead maintain the infection in a latent state. These individuals generally become positive in response to a tuberculin skin test and may develop clinical TB at a later date, particularly if their immune systems are compromised. Latently infected individuals are interesting for two reasons. First, they are an important reservoir of M. tuberculosis, which needs to be considered for TB control. Second, if detected prior to recrudescence of the disease, they represent a human population that is making a protective immune response to M. tuberculosis, which is very important for defining correlates of protective immunity. In this study, we show that while responsiveness to early secretory antigenic target 6 is a good marker for M. tuberculosis infection, a strong response to the 16-kDa Rv2031c antigen (HspX or alpha-crystallin) is largely restricted to latently infected individuals, offering the possibility of differential immunodiagnosis of, or therapeutic vaccination against, TB.

Quantitative T cell assay reflects infectious load of Mycobacterium tuberculosis in an endemic case contact model.

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.

Increased expression of mRNA encoding interleukin (IL)-4 and its splice variant IL-4delta2 in cells from contacts of Mycobacterium tuberculosis, in the absence of in vitro stimulation.

Expression of interleukin (IL)-4 is increased in tuberculosis and thought to be detrimental. We show here that in healthy contacts there is increased expression of its naturally occurring antagonist, IL-4delta2 (IL-4delta2). We identified contacts by showing that their peripheral blood mononuclear cells (PBMC) released interferon (IFN)-gamma in response to the Mycobacterium tuberculosis-specific antigen 6 kDa early secretory antigenic target (ESAT-6). Fresh unstimulated PBMC from these contacts contained higher levels of mRNA encoding IL-4delta2 (P=0.002) than did cells from ESAT-6 negative donors (noncontacts). These data indicate that contact with M. tuberculosis induces unusual, previously unrecognized, immunological events. We tentatively hypothesize that progression to active disease might depend upon the underlying ratio of IL-4 to IL-4delta2.