Philip C. Hill

Genome-wide association analyses identifies a susceptibility locus for tuberculosis on chromosome 18q11.2

We combined two tuberculosis genome-wide association studies from Ghana and The Gambia with subsequent replication in a combined 11,425 individuals. rs4331426, located in a gene-poor region on chromosome 18q11.2, was associated with disease (combined P = 6.8 × 10−9, odds ratio = 1.19, 95% CI = 1.13–1.27). Our study demonstrates that genome-wide association studies can identify new susceptibility loci for infectious diseases, even in African populations, in which levels of linkage disequilibrium are particularly low.

Progression to Active Tuberculosis, but Not Transmission, Varies by Mycobacterium tuberculosis Lineage in The Gambia

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.

Recognition of Stage-Specific Mycobacterial Antigens Differentiates between Acute and Latent Infections with Mycobacterium tuberculosis

ABSTRACT 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 α-crystallin) is largely restricted to latently infected individuals, offering the possibility of differential immunodiagnosis of, or therapeutic vaccination against, TB.

Pattern and diversity of cytokine production differentiates between Mycobacterium tuberculosis infection and disease.

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‐γ+TNF‐α+IL‐2+CD4+T cells compared with contacts. TB cases also had a significantly higher proportion of cells single‐positive for TNF‐α, 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‐γ 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.

Large-Scale Evaluation of Enzyme-Linked Immunospot Assay and Skin Test for Diagnosis of Mycobacterium tuberculosis Infection against a Gradient of Exposure in The Gambia

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.

DC-SIGN (CD209), pentraxin 3 and vitamin D receptor gene variants associate with pulmonary tuberculosis risk in West Africans.

We investigated the role of DC-SIGN (CD209), long pentraxin 3 (PTX3) and vitamin D receptor (VDR) gene single nucleotide polymorphisms (SNPs) in susceptibility to pulmonary tuberculosis (TB) in 321 TB cases and 347 healthy controls from Guinea-Bissau. Five additional, functionally relevant SNPs within toll-like receptors (TLRs) 2, 4 and 9 were typed but found, when polymorphic, not to affect host vulnerability to pulmonary TB. We did not replicate an association between SNPs in the DC-SIGN promoter and TB. However, we found that two polymorphisms, one in DC-SIGN and one in VDR, were associated in a nonadditive model with disease risk when analyzed in combination with ethnicity (P=0.03 for DC-SIGN and P=0.003 for VDR). In addition, PTX3 haplotype frequencies significantly differed in cases compared to controls and a protective effect was found in association with a specific haplotype (OR 0.78, 95% CI 0.63–0.98). Our findings support previous data showing that VDR SNPs modulate the risk for TB in West Africans and suggest that variation within DC-SIGN and PTX3 also affect the disease outcome.

Longitudinal Assessment of an ELISPOT Test for Mycobacterium tuberculosis Infection

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.

Nasopharyngeal Carriage of Streptococcus pneumoniae in Gambian Villagers

BACKGROUND To prepare for the introduction of a pneumococcal conjugate vaccine of restricted valency, we studied the nasopharyngeal carriage of Streptococcus pneumoniae in Gambian villagers. METHODS A cross-sectional survey was conducted in 21 villages after a census. We recorded demographic characteristics, information on medical history, and data on possible risk factors for carriage from subjects. We collected a nasopharyngeal swab specimen from each subject for isolation and serotyping of S. pneumoniae and for antibiotic susceptibility testing. RESULTS The prevalence of S. pneumoniae carriage among 2872 villagers was 72%. It was highest among infants (i.e., children aged <1 year; 97%); the rate was 93% among babies aged <1 month and decreased with increasing age (P<.001). Prevalence of carriage was linked to proximity to another village. Sixty-three percent of isolates recovered from children aged <5 years were covered by the 7-valent vaccine or were of a vaccine-related serotype, compared with 43% of isolates overall. Forty-three isolates (14.3%) tested were initially penicillin resistant; none had high-level resistance, and 4 had intermediate resistance. The rates of resistance to other antibiotics were as follows: trimethoprim-sulfamethoxazole, 39%; tetracycline, 32.3%; chloramphenicol, 6.3%; cefotaxime, 0.3%; and erythromycin, 0%. The rates were highest for isolates of vaccine serotypes. CONCLUSIONS Pneumococcal carriage rates among Gambian villagers are very high. A pneumococcal conjugate vaccine of restricted valency should reduce the pool of antibiotic-resistant pneumococci. The large reservoir of pneumococci of nonvaccine serotypes will require close monitoring when the vaccine is introduced.

Incidence of Tuberculosis and the Predictive Value of ELISPOT and Mantoux Tests in Gambian Case Contacts

Background Studies of Tuberculosis (TB) case contacts are increasingly being utilised for understanding the relationship between M. tuberculosis and the human host and for assessing new interventions and diagnostic tests. We aimed to identify the incidence rate of new TB cases among TB contacts and to relate this to their initial Mantoux and ELISPOT test results. Methods and Findings After initial Mantoux and ELISPOT tests and exclusion of co-prevalent TB cases, we followed 2348 household contacts of sputum smear positive TB cases. We visited them at 3 months, 6 months, 12 months, 18 months and 24 months, and investigated those with symptoms consistent with TB. Those who were diagnosed separately at a government clinic had a chest x-ray. Twenty six contacts were diagnosed with definite TB over 4312 person years of follow-up (Incidence rate 603/100,000 person years; 95% Confidence Interval, 370–830). Nine index and secondary case pairs had cultured isolates available for genotyping. Of these, 6 pairs were concordant and 3 were discordant. 2.5% of non-progressors were HIV positive compared to 12% of progressors (HR 6.2; 95% CI 1.7–22.5; p = 0.010). 25 secondary cases had initial Mantoux results, 14 (56%) were positive ; 21 had initial ELISPOT results, 11 (52%) were positive; 15 (71%) of 21 tested were positive by one or the other test. Of the 6 contacts who had concordant isolates with their respective index case, 4 (67%) were Mantoux positive at recruitment, 3 (50%) were ELISPOT positive; 5 (83%) were positive by one or other of the two tests. ELISPOT positive contacts, and those with discordant results, had a similar rate of progression to those who were Mantoux positive. Those negative on either or both tests had the lowest rate of progression. Conclusions The incidence rate of TB disease in Gambian TB case contacts, after screening for co-prevalent cases, was 603/100,000 person years. Since initial ELISPOT test and Mantoux tests were each positive in only just over half of cases, but 71% were positive by one or other test, positivity by either might be the best indication for preventive treatment. These data do not support the replacement of the Mantoux test by an ELISPOT test in The Gambia or similar settings.

Nasopharyngeal Carriage of Streptococcus pneumoniae in Gambian Infants: A Longitudinal Study

BACKGROUND To prepare for national introduction of a pneumococcal conjugate vaccine of restricted valency, we studied nasopharyngeal carriage of Streptococcus pneumoniae in Gambian infants. METHODS We studied 236 infants in 21 villages. We collected nasopharyngeal swab samples at birth, twice per month for 6 months, and every second month until 1 year of age. We studied time to acquisition and duration of pneumococcal carriage according to serotype. RESULTS All infants carried S. pneumoniae at some point. Sixty-five serotypes were found, and the 5 most common serotypes (6B, 19F, 6A, 14, and 23F) accounted for 51% of isolates. The mean age at first acquisition of carriage was 33 days (95% confidence interval, 29-36 days). There were no significant differences in acquisition rates between the 6 most common serotypes (P = .067) or between vaccine serotypes, vaccine-related serotypes, or nonvaccine serotypes (P = .317). However, the duration of carriage differed significantly between the 6 most common serotypes (P = .004). The rate of reacquisition of carriage and the duration of carriage did not differ significantly between the 6 most common serotypes (P = .229 and P = .669 respectively). However, nonvaccine types were acquired faster (P = .004) and were carried for a shorter duration (P < .001) than were vaccine serotypes. A previous episode of serotype 14 carriage was associated with delayed reacquisition of this serotype (P = .005) and longer duration of carriage (P = .017). CONCLUSIONS The data provided in this study regarding time to acquisition and duration of pneumococcal carriage in Gambian infants provide an important baseline for evaluating the impact of the introduction of a pneumococcal conjugate vaccine in The Gambia and elsewhere in Africa.