Correction: Discovery and validation of a prognostic proteomic signature for tuberculosis progression: A prospective cohort study.

[This corrects the article DOI: 10.1371/journal.pmed.1002781.].

Discovery and validation of a prognostic proteomic signature for tuberculosis progression: A prospective cohort study.

BACKGROUND: A nonsputum blood test capable of predicting progression of healthy individuals to active tuberculosis (TB) before clinical symptoms manifest would allow targeted treatment to curb transmission. We aimed to develop a proteomic biomarker of risk of TB progression for ultimate translation into a point-of-care diagnostic. METHODS AND FINDINGS: Proteomic TB risk signatures were discovered in a longitudinal cohort of 6,363 Mycobacterium tuberculosis-infected, HIV-negative South African adolescents aged 12-18 years (68% female) who participated in the Adolescent Cohort Study (ACS) between July 6, 2005 and April 23, 2007, through either active (every 6 months) or passive follow-up over 2 years. Forty-six individuals developed microbiologically confirmed TB disease within 2 years of follow-up and were selected as progressors; 106 nonprogressors, who remained healthy, were matched to progressors. Over 3,000 human proteins were quantified in plasma with a highly multiplexed proteomic assay (SOMAscan). Three hundred sixty-one proteins of differential abundance between progressors and nonprogressors were identified. A 5-protein signature, TB Risk Model 5 (TRM5), was discovered in the ACS training set and verified by blind prediction in the ACS test set. Poor performance on samples 13-24 months before TB diagnosis motivated discovery of a second 3-protein signature, 3-protein pair-ratio (3PR) developed using an orthogonal strategy on the full ACS subcohort. Prognostic performance of both signatures was validated in an independent cohort of 1,948 HIV-negative household TB contacts from The Gambia (aged 15-60 years, 66% female), longitudinally followed up for 2 years between March 5, 2007 and October 21, 2010, sampled at baseline, month 6, and month 18. Amongst these contacts, 34 individuals progressed to microbiologically confirmed TB disease and were included as progressors, and 115 nonprogressors were included as controls. Prognostic performance of the TRM5 signature in the ACS training set was excellent within 6 months of TB diagnosis (area under the receiver operating characteristic curve [AUC] 0.96 [95% confidence interval, 0.93-0.99]) and 6-12 months (AUC 0.76 [0.65-0.87]) before TB diagnosis. TRM5 validated with an AUC of 0.66 (0.56-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. The 3PR signature yielded an AUC of 0.89 (0.84-0.95) within 6 months of TB diagnosis and 0.72 (0.64-0.81) 7-12 months before TB diagnosis in the entire South African discovery cohort and validated with an AUC of 0.65 (0.55-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. Signature validation may have been limited by a systematic shift in signal magnitudes generated by differences between the validation assay when compared to the discovery assay. Further validation, especially in cohorts from non-African countries, is necessary to determine how generalizable signature performance is. CONCLUSIONS: Both proteomic TB risk signatures predicted progression to incident TB within a year of diagnosis. To our knowledge, these are the first validated prognostic proteomic signatures. Neither meet the minimum criteria as defined in the WHO Target Product Profile for a progression test. More work is required to develop such a test for practical identification of individuals for investigation of incipient, subclinical, or active TB disease for appropriate treatment and care.

Discovery and Validation of a Six-Marker Serum Protein Signature for the Diagnosis of Active Pulmonary Tuberculosis.

New non-sputum biomarker tests for active tuberculosis (TB) diagnostics are of the highest priority for global TB control. We performed in-depth proteomic analysis using the 4,000-plex SOMAscan assay on 1,470 serum samples from seven countries where TB is endemic. All samples were from patients with symptoms and signs suggestive of active pulmonary TB that were systematically confirmed or ruled out for TB by culture and clinical follow-up. HIV coinfection was present in 34% of samples, and 25% were sputum smear negative. Serum protein biomarkers were identified by stability selection using L1-regularized logistic regression and by Kolmogorov-Smirnov (KS) statistics. A naive Bayes classifier using six host response markers (HR6 model), including SYWC, kallistatin, complement C9, gelsolin, testican-2, and aldolase C, performed well in a training set (area under the sensitivity-specificity curve [AUC] of 0.94) and in a blinded verification set (AUC of 0.92) to distinguish TB and non-TB samples. Differential expression was also highly significant (P < 10-20) for previously described TB markers, such as IP-10, LBP, FCG3B, and TSP4, and for many novel proteins not previously associated with TB. Proteins with the largest median fold changes were SAA (serum amyloid protein A), NPS-PLA2 (secreted phospholipase A2), and CA6 (carbonic anhydrase 6). Target product profiles (TPPs) for a non-sputum biomarker test to diagnose active TB for treatment initiation (TPP#1) and for a community-based triage or referral test (TPP#2) have been published by the WHO. With 90% sensitivity and 80% specificity, the HR6 model fell short of TPP#1 but reached TPP#2 performance criteria. In conclusion, we identified and validated a six-marker signature for active TB that warrants diagnostic development on a patient-near platform.

Highly Multiplexed Proteomic Analysis of Quantiferon Supernatants To Identify Biomarkers of Latent Tuberculosis Infection.

The tests for diagnosing latent tuberculosis infection (LTBI) are limited by a poor predictive value for identifying people at the highest risk for progressing to active tuberculosis (TB) and have various sensitivities and specificities in different populations. Identifying a more robust signature for LTBI is important for TB prevention and elimination. A pilot study was conducted with samples from immigrants to the United States that were screened for LTBI by the three commercially approved tests, namely, the tuberculin skin test (TST), the Quantiferon-TB Gold in-tube (QFT-GIT), and the T-SPOT.TB (T-SPOT). QFT-GIT supernatants from 13 people with concordant positive results and 26 people with concordant negative results were analyzed via the highly multiplexed SOMAscan proteomic assay. The proteins in the stimulated supernatants that distinguished LTBI from controls included interleukin-2 (IL-2), monocyte chemotactic protein 2 (MCP-2), interferon gamma inducible protein-10 (IP-10), interferon gamma (IFN-γ), tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT), monokine induced by gamma interferon (MIG), and granzyme B (P <0.00001). In addition, antigen stimulation increased the expression of heparin-binding EGF-like growth factor (HB-EGF) and activin AB in LTBI samples. In nil tubes, LIGHT was the most significant marker (P <0.0001) and was elevated in LTBI subjects. Other prominent markers in nonstimulated QFT-GIT supernatants were the complement-3 components C3b, iC3b, and C3d, which were upregulated in LTBI and markedly decreased upon stimulation. We found known and novel proteins that warrant further studies for developing improved tests for LTBI, for predicting progression to active disease, and for discriminating LTBI from active TB.

Acceptability of interferon-gamma release assays among healthcare workers who receive routine employee tuberculosis testing.

BACKGROUND: Healthcare workers (HCWs) undergo annual testing for latent tuberculosis infection (LTBI). OBJECTIVE: Compare acceptability of tuberculin skin test (TST) and interferon-gamma release assay (IGRA) among HCWs. METHODS: HCWs at four medical centers in the US were administered an acceptability questionnaire including a brief objective description of both tests and eliciting attitudes regarding TST and IGRAs, confidence in results, and likelihood of taking LTBI treatment. RESULTS: Of 406 participants, 75% had never heard of IGRAs. IGRAs were preferred to TST. Belief in accuracy of hypothetical positive results of TST or IGRA and willingness to accept LTBI treatment were similar across tests. When presented with hypothetical discordant results, HCWs expressed more confidence in IGRAs. Perceived accuracy of results was the most important factor in test preferences. CONCLUSIONS: Although HCWs preferred and indicated more confidence in IGRAs, the likelihood that HCWs would believe LTBI diagnosis and initiate treatment based on positive results was similar for TST and IGRAs.

Proteomic Profiles in Advanced Age-Related Macular Degeneration Using an Aptamer-Based Proteomic Technology.

PURPOSE: To explore top-ranked plasma proteins related to neovascular age-related macular degeneration (AMD) and geographic atrophy (GA), and explore pathways related to neovascular AMD and GA. METHODS: We conducted a pilot study of patients with neovascular AMD (n = 10), GA (n = 10), and age-matched cataract controls (n = 10) who were recruited into an AMD registry. We measured 4001 proteins in ethylenediaminetetraacetic acid plasma samples using an aptamer-based proteomic technology. Relative concentrations of each of 4001 proteins were log (base 2) transformed and compared between cases of neovascular AMD and GA versus controls using linear regression. Pathway analysis was conducted using pathways downloaded from Reactome. RESULTS: In this pilot study, higher levels of vinculin and lower levels of CD177 were found in patients with neovascular AMD compared with controls. Neuregulin-4 was higher and soluble intercellular adhesion molecule-1 was lower in patients with GA compared with controls. For neovascular AMD, cargo trafficking to the periciliary membrane, fibroblast growth factor receptor 3b ligand binding and activation, and vascular endothelial growth factor-related pathways were in the top ranked pathways. The top-ranked pathways for GA included several related to ErbB4 signaling. CONCLUSIONS: We found different proteins and different pathways associated with neovascular AMD and GA. Vinculin and some of the top-ranked pathways have been previously associated with AMD, whereas others have not been described. TRANSLATIONAL RELEVANCE: Biomarkers identified in plasma likely reflect systemic alterations in protein expression and may improve our understanding of the mechanisms leading to AMD.

Transnational Record Linkage for Tuberculosis Surveillance and Program Evaluation.

OBJECTIVE: Pre-immigration tuberculosis (TB) screening, followed by post-arrival rescreening during the first year, is critical to reducing TB among foreign-born people in the United States. However, existing U.S. public health surveillance is inadequate to monitor TB among immigrants during subsequent years. We developed and tested a novel method for ascertaining post-U.S.-arrival TB outcomes among high-TB-risk immigrant cohorts to improve surveillance. METHODS: We used a probabilistic record linkage program to link pre-immigration screening records from U.S.-bound immigrants from the Philippines (n=422,593) and Vietnam (n=214,401) with the California TB registry during 2000-2010. We estimated sensitivity using Monte Carlo simulations to account for uncertainty in key inputs. Specificity was evaluated by using a time-stratified approach, which defined false-positives as TB records linked to pre-immigration screening records dated after the person had arrived in the United States. RESULTS: TB was reported in 4,382 and 2,830 people born in the Philippines and Vietnam, respectively, in California during the study period. Of these TB cases, records for 973 and 452 cases of people born in the Philippines and Vietnam, respectively, were linked to pre-immigration screening records. Sensitivity and specificity of linkage were 89% (90% credible interval [CrI] 83, 97) and 100%, respectively, for the Philippines, and 90% (90% CrI 83, 98) and 99.9%, respectively, for Vietnam. CONCLUSION: Electronic linkage of pre-immigration screening records to a domestic TB registry was feasible, sensitive, and highly specific in two high-priority immigrant cohorts. Transnational record linkage can be used for program evaluation and routine monitoring of post-U.S.-arrival TB risk among immigrants, but requires interagency data sharing and collaboration.

Epidemiology of tuberculosis in young children in the United States.

OBJECTIVES: To estimate tuberculosis (TB) rates among young children in the United States by children's and parents' birth origins and describe the epidemiology of TB among young children who are foreign-born or have at least 1 foreign-born parent. METHODS: Study subjects were children <5 years old diagnosed with TB in 20 US jurisdictions during 2005-2006. TB rates were calculated from jurisdictions' TB case counts and American Community Survey population estimates. An observational study collected demographics, immigration and travel histories, and clinical and source case details from parental interviews and health department and TB surveillance records. RESULTS: Compared with TB rates among US-born children with US-born parents, rates were 32 times higher in foreign-born children and 6 times higher in US-born children with foreign-born parents. Most TB cases (53%) were among the 29% of children who were US born with foreign-born parents. In the observational study, US-born children with foreign-born parents were more likely than foreign-born children to be infants (30% vs. 7%), Hispanic (73% vs. 37%), diagnosed through contact tracing (40% vs. 7%), and have an identified source case (61% vs. 19%); two-thirds of children were exposed in the United States. CONCLUSIONS: Young children who are US born of foreign-born parents have relatively high rates of TB and account for most cases in this age group. Prompt diagnosis and treatment of adult source cases, effective contact investigations prioritizing young contacts, and targeted testing and treatment of latent TB infection are necessary to reduce TB morbidity in this population.

Tuberculosis screening by tuberculosis skin test or QuantiFERON-TB Gold In-Tube Assay among an immigrant population with a high prevalence of tuberculosis and BCG vaccination.

RATIONALE: Each year 1 million persons acquire permanent U.S. residency visas after tuberculosis (TB) screening. Most applicants undergo a 2-stage screening with tuberculin skin test (TST) followed by CXR only if TST-positive at > 5 mm. Due to cross reaction with bacillus Calmette-Guérin (BCG), TST may yield false positive results in BCG-vaccinated persons. Interferon gamma release assays exclude antigens found in BCG. In Vietnam, like most high TB-prevalence countries, there is universal BCG vaccination at birth. OBJECTIVES: 1. Compare the sensitivity of QuantiFERON-TB Gold In-Tube Assay (QFT) and TST for culture-positive pulmonary TB. 2. Compare the age-specific and overall prevalence of positive TST and QFT among applicants with normal and abnormal CXR. METHODS: We obtained TST and QFT results on 996 applicants with abnormal CXR, of whom 132 had TB, and 479 with normal CXR. RESULTS: The sensitivity for tuberculosis was 86.4% for QFT; 89.4%, 81.1%, and 52.3% for TST at 5, 10, and 15 mm. The estimated prevalence of positive results at age 15-19 years was 22% and 42% for QFT and TST at 10 mm, respectively. The prevalence increased thereafter by 0.7% year of age for TST and 2.1% for QFT, the latter being more consistent with the increase in TB among applicants. CONCLUSIONS: During 2-stage screening, QFT is as sensitive as TST in detecting TB with fewer requiring CXR and being diagnosed with LTBI. These data support the use of QFT over TST in this population.

Latent TB infection treatment acceptance and completion in the United States and Canada.

BACKGROUND: Treatment of latent TB infection (LTBI) is essential for preventing TB in North America, but acceptance and completion of this treatment have not been systematically assessed. METHODS: We performed a retrospective, randomized two-stage cross-sectional survey of treatment and completion of LTBI at public and private clinics in 19 regions of the United States and Canada in 2002. RESULTS: At 32 clinics that both performed tuberculin skin testing and offered treatment, 123 (17.1%; 95% CI, 14.5%-20.0%) of 720 subjects tested and offered treatment declined. Employees at health-care facilities were more likely to decline (odds ratio [OR], 4.74; 95% CI, 1.75-12.9; P = .003), whereas those in contact with a patient with TB were less likely to decline (OR, 0.19; 95% CI, 0.07-0.50; P = .001). At 68 clinics starting treatment regardless of where skin testing was performed, 1,045 (52.7%; 95% CI, 48.5%-56.8%) of 1,994 people starting treatment failed to complete the recommended course. Risk factors for failure to complete included starting the 9-month isoniazid regimen (OR, 2.08; 95% CI, 1.23-3.57), residence in a congregate setting (nursing home, shelter, or jail; OR, 2.94; 95% CI, 1.58-5.56), injection drug use (OR, 2.13; 95% CI, 1.04-4.35), age >or= 15 years (OR, 1.49; 95% CI, 1.14-1.94), and employment at a health-care facility (1.37; 95% CI, 1.00-1.85). CONCLUSIONS: Fewer than half of the people starting treatment of LTBI completed therapy. Shorter regimens and interventions targeting residents of congregate settings, injection drug users, and employees of health-care facilities are needed to increase completion.