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Background: Increasing the diversity of plastic surgery trainees is an important step in providing optimal care for our increasingly diverse patient populations. Given that information presented on residency programs' websites can strongly influence applicants' decisions to apply to or rank a program, demonstrating a commitment to diversity, equity, and inclusion (DEI) on program websites may aid in recruiting applicants with URM background., Methods: Using 8 DEI-related criteria, we evaluated the websites of 103 plastic surgery residency programs for the presence of DEI-related content during the month of June 2022. Each program was evaluated by 2 individual graders. We analyzed the data with confirmatory factor analysis in R using the Lavaan package., Results: On average, programs fulfilled 2.1 ± 1.6 of the metrics with a range of 0-7 fulfilled per program. Our model revealed that the criteria were a high-quality (P < 0.0001) measure of DEI-related metrics. There was a significant association between program size and presence of DEI-related criteria (linear 0.039; quadratic -0.005; both P < 0.01), such that mid-sized programs (16-18 residents) had the highest quality of DEI advertising compared to both small and large programs. Programs associated with a USNWR Top 20 Hospital were less likely to achieve high level of DEI-related criteria than other programs (P < 0.0001)., Conclusion: Mid-sized programs had greater DEI quality on their websites, while smaller and larger programs similarly had poorer assessed quality. There is room for all programs to improve the presence of DEI-related material on their websites, especially related to care of transgender populations., Competing Interests: Conflicts of interest and sources of funding: No funds were received for this study. The authors have no conflicts of interest to declare., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The diagnosis of paediatric tuberculosis remains a challenge due to the non-specificity of symptoms and the paucibacillary nature of tuberculosis in children. However, in the development of new tuberculosis diagnostics, the unique needs of children and adolescents are rarely considered in the design process, with delays in evaluation and approval. No clear guidance is available on when and how to include children and adolescents in tuberculosis diagnostic development and evaluation. To address this gap, we conducted a Delphi consensus process with 42 stakeholders, including one qualitative and two quantitative rounds. Consensus was achieved on 20 statements, with agreement that the needs and perspectives of children, adolescents, and their caregivers should be incorporated throughout diagnostic design and evaluation. Opportunities exist for the early use of well characterised samples and prospective enrolment of children and adolescents in tuberculosis diagnostic evaluation, with consideration of the type of test, expected benefit, and potential risks. Pathogen-based tests might be initially optimised and assessed in adults and adolescents, but parallel evaluation in children is needed for host-based tests. Late-stage evaluation and implementation studies should examine combination testing and integration into clinical algorithms. The statements support collaboration between developers, researchers, regulators, and users to widen and accelerate the diagnostic pipeline for paediatric tuberculosis., Competing Interests: Declaration of interests SL receives royalties from UpToDate on the tuberculosis in Pregnancy topic; and receives funding from Merck for her institution for investigator-initiated SARS-CoV-2 related research. EMB and LO are supported by a fellowship from the European Society for Paediatric Infectious Diseases. ELM is supported by a Canadian Institutes of Health Research Fellowship (472823). MPN is supported by an Australian National Health and Medical Research Council Investigator Award 1174455. HJZ is supported by the South African Medical Research Council. All other authors declare no competing interests. This consensus process was supported by the National Institute of Allergy and Infectious Diseases Feasibility of Novel Diagnostics for TB in Endemic Countries for TB studies (U01AI152087, U01AI152084, and U01AI152075)., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Antibody features vary with tuberculosis (TB) disease state. Whether clinical variables, such as age or sex, influence associations between Mycobacterium tuberculosis-specific antibody responses and disease state is not well explored. Here we profiled Mycobacterium tuberculosis-specific antibody responses in 140 TB-exposed South African individuals from the Adolescent Cohort Study. We identified distinct response features in individuals progressing to active TB from non-progressing, matched controls. A multivariate antibody score differentially associated with progression (SeroScore) identified progressors up to 2 years before TB diagnosis, earlier than that achieved with the RISK6 transcriptional signature of progression. We validated these antibody response features in the Grand Challenges 6-74 cohort. Both the SeroScore and RISK6 correlated better with risk of TB progression in adolescents compared with adults, and in males compared with females. This suggests that age and sex are important, underappreciated modifiers of antibody responses associated with TB progression., (© 2024. The Author(s).)

Background: Improved monitoring of Mycobacterium tuberculosis response to treatment is urgently required. We previously developed the molecular bacterial load assay (MBLA), but it is challenging to integrate into the clinical diagnostic laboratory due to a labor-intensive protocol required at biosafety level 3 (BSL-3). A modified assay was needed., Methods: The rapid enumeration and diagnostic for tuberculosis (READ-TB) assay was developed. Acetic acid was tested and compared to 4 M guanidine thiocyanate to be simultaneously bactericidal and preserve mycobacterial RNA. The extraction was based on silica column technology and incorporated low-cost reagents: 3 M sodium acetate and ethanol for the RNA extraction to replace phenol-chloroform. READ-TB was fully validated and compared directly to the MBLA using sputa collected from individuals with tuberculosis., Results: Acetic acid was bactericidal to M. tuberculosis with no significant loss in 16S rRNA or an unprotected mRNA fragment when sputum was stored in acetic acid at 25°C for 2 weeks or -20°C for 1 year. This novel use of acetic acid allows processing of sputum for READ-TB at biosafety level 2 (BSL-2) on sample receipt. READ-TB is semiautomated and rapid. READ-TB correlated with the MBLA when 85 human sputum samples were directly compared (R2 = 0.74)., Conclusions: READ-TB is an improved version of the MBLA and is available to be adopted by clinical microbiology laboratories as a tool for tuberculosis treatment monitoring. READ-TB will have a particular impact in low- and middle-income countries (LMICs) for laboratories with no BSL-3 laboratory and for clinical trials testing new combinations of anti-tuberculosis drugs., (© Association for Diagnostics & Laboratory Medicine 2024.)

Activin A strongly influences immune responses; yet, few studies have examined its role in infectious diseases. We measured serum activin A levels in two independent tuberculosis (TB) patient cohorts and in patients with pneumonia and sarcoidosis. Serum activin A levels were increased in TB patients compared to healthy controls, including those with positive tuberculin skin tests, and paralleled severity of disease, assessed by X-ray scores. In pneumonia patients, serum activin A levels were also raised, but in sarcoidosis patients, levels were lower. To determine whether blockade of the activin A signaling axis could play a functional role in TB, we harnessed a soluble activin type IIB receptor fused to human IgG1 Fc, ActRIIB-Fc, as a ligand trap in a murine TB model. The administration of ActRIIB-Fc to Mycobacterium tuberculosis -infected mice resulted in decreased bacterial loads and increased numbers of CD4 effector T cells and tissue-resident memory T cells in the lung. Increased frequencies of tissue-resident memory T cells corresponded with downregulated T-bet expression in lung CD4 and CD8 T cells. Altogether, the results suggest a disease-exacerbating role of ActRIIB signaling pathways. Serum activin A may be useful as a biomarker for diagnostic triage of active TB or monitoring of anti-tuberculosis therapy., Importance: Tuberculosis remains the leading cause of death by a bacterial pathogen. The etiologic agent of tuberculosis, Mycobacterium tuberculosis , can remain dormant in the infected host for years before causing disease. Significant effort has been made to identify biomarkers that can discriminate between latently infected and actively diseased individuals. We found that serum levels of the cytokine activin A were associated with increased lung pathology and could discriminate between active tuberculosis and tuberculin skin-test-positive healthy controls. Activin A signals through the ActRIIB receptor, which can be blocked by administration of the ligand trap ActRIIB-Fc, a soluble activin type IIB receptor fused to human IgG1 Fc. In a murine model of tuberculosis, we found that ActRIIB-Fc treatment reduced mycobacterial loads. Strikingly, ActRIIB-Fc treatment significantly increased the number of tissue-resident memory T cells. These results suggest a role for ActRIIB signaling pathways in host responses to Mycobacterium tuberculosis and activin A as a biomarker of ongoing disease., Competing Interests: The authors declare no conflict of interest.

Background: Tuberculosis (TB) and COVID-19 are the two leading causes of infectious disease mortality worldwide, and their overlap is likely frequent and inevitable. Previous research has shown increased mortality in TB/COVID-coinfected individuals, and emerging evidence suggests that COVID-19 may increase susceptibility to TB. However, the immunological mechanisms underlying these interactions remain unclear. In this study, we aimed to elucidate the impact of prior or concurrent COVID-19 infection on immune profiles of TB patients and those with other respiratory diseases (ORD)., Methods: Serum and nasopharyngeal samples were collected from 161 Gambian adolescents and adults with either TB or an ORD. Concurrent COVID-19 infection was determined by PCR, while prior COVID-19 was defined by antibody seropositivity. Multiplex cytokine immunoassays were used to quantify 27 cytokines and chemokines in patient serum samples at baseline, and throughout treatment in TB patients., Results: Strikingly, TB and ORD patients with prior COVID-19 infection were found to have significantly reduced expression of several cytokines, including IL-1β, TNF-α and IL-7, compared to those without (p<0.035). Moreover, at month-six of anti-TB treatment, seropositive patients had lower serum Basic FGF (p=0.0115), IL-1β (p=0.0326) and IL-8 (p=0.0021) than seronegative. TB patients with acute COVID-19 coinfection had lower levels of IL-8, IL-13, TNF-α and IP-10 than TB-only patients, though these trends did not reach significance (p>0.035)., Conclusions: Our findings demonstrate that COVID-19 infection alters the subsequent response to TB and ORDs, potentially contributing to pathogenesis. Further work is necessary to determine whether COVID-19 infection accelerates TB disease progression, though our results experimentally support this hypothesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Cottam, Manneh, Gindeh, Sillah, Cham, Mendy, Barry, Coker, Daffeh, Badjie, Barry, Owolabi, Winter, Walzl and Sutherland.)

Background: Inclusion of Histoplasma in the World Health Organization's first Fungal Priority Pathogens List under "high-priority" fungal species highlights the need for robust surveillance of Histoplasma spp. in endemic and underrepresented regions. Despite increasing reports of histoplasmosis in Africa, data on the burden of this fungal disease are sparse in The Gambia. This baseline study examined the human seroprevalence of anti- Histoplasma antibody in a TB patient group in The Gambia, explored associations between seropositivity and demographic and clinical variables, and proposes future research directions., Methods: Biobanked plasma samples were selected from active TB cases with variable HIV infection status. Latex agglutination tests were performed on samples from 52 study participants to detect the presence of anti- Histoplasma antibodies. Potential risk factors for Histoplasma exposure were explored using logistic regression analysis., Results: The sample seroprevalence of anti- Histoplasma antibody was 28.8% (n = 15/52; 95% CI, 17.1%-43.1%). Multivariable logistic regression analysis identified a statistically significant association between Histoplasma seropositivity and age (odds ratio, 0.91; 95% CI, 0.84-0.98; P = .008)., Conclusions: This baseline study provides evidence of Histoplasma seropositivity in TB patients in The Gambia and explores risk factors for exposure. The small sample size and use of the LAT in TB and HIV-positive patient groups are significant study limitations. Future research directions are proposed to ascertain the burden of Histoplasma in general and patient populations and explore the context-specific risk factors for exposure and infection in The Gambia., Competing Interests: Potential conflicts of interest. All authors: no reported conflicts., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Background: Halting transmission of Mycobacterium tuberculosis (Mtb) by identifying infectious individuals early is key to eradicating tuberculosis (TB). Here we evaluate face mask sampling as a tool for stratifying the infection risk of individuals with pulmonary TB (PTB) to their household contacts., Methods: Forty-six sputum-positive PTB patients in The Gambia (August 2016-November 2017) consented to mask sampling prior to commencing treatment. Incident Mtb infection was defined in 181 of their 217 household contacts as QuantiFERON conversion or an increase in interferon-γ of ≥1 IU/mL, 6 months after index diagnosis. Multilevel mixed-effects logistical regression analysis with cluster adjustment by household was used to identify predictors of incident infection., Results: Mtb was detected in 91% of PTB mask samples with high variation in IS6110 copies (5.3 × 102 to 1.2 × 107). A high mask Mtb level (≥20 000 IS6110 copies) was observed in 45% of cases and was independently associated with increased likelihood of incident Mtb infection in contacts (adjusted odds ratio, 3.20 [95% confidence interval, 1.26-8.12]; P = .01), compared with cases having low-positive/negative mask Mtb levels. Mask Mtb level was a better predictor of incident Mtb infection than sputum bacillary load, chest radiographic characteristics, or sleeping proximity., Conclusions: Mask sampling offers a sensitive and noninvasive tool to support the stratification of individuals who are most infectious in high-TB-burden settings. Our approach can provide better insight into community transmission in complex environments., Competing Interests: Potential conflicts of interest. M. P. has been a consultant to Qiagen in the last 36 months for unrelated work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

Mycobacterium tuberculosis (M.tb) causes tuberculosis (TB) and remains one of the leading causes of mortality due to an infectious pathogen. Host immune responses have been implicated in driving the progression from infection to severe lung disease. We analyzed longitudinal RNA sequencing (RNAseq) data from the whole blood of 74 TB progressors whose samples were grouped into four six-month intervals preceding diagnosis (the GC6-74 study). We additionally analyzed RNAseq data from an independent cohort of 90 TB patients with positron emission tomography-computed tomography (PET-CT) scan results which were used to categorize them into groups with high and low levels of lung damage (the Catalysis TB Biomarker study). These groups were compared to non-TB controls to obtain a complete whole blood transcriptional profile for individuals spanning from early stages of M.tb infection to TB diagnosis. The results revealed a steady increase in the number of genes that were differentially expressed in progressors at time points closer to diagnosis with 278 genes at 13-18 months, 742 at 7-12 months and 5,131 detected 1-6 months before diagnosis and 9,205 detected in TB patients. A total of 2,144 differentially expressed genes were detected when comparing TB patients with high and low levels of lung damage. There was a large overlap in the genes upregulated in progressors 1-6 months before diagnosis (86%) with those in TB patients. A comprehensive pathway analysis revealed a potent activation of neutrophil and platelet mediated defenses including neutrophil and platelet degranulation, and NET formation at both time points. These pathways were also enriched in TB patients with high levels of lung damage compared to those with low. These findings suggest that neutrophils and platelets play a critical role in TB pathogenesis, and provide details of the timing of specific effector mechanisms that may contribute to TB lung pathology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Meier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The development of a fast and accurate, non-sputum-based point-of-care triage test for tuberculosis (TB) would have a major impact on combating the TB burden worldwide. A new fingerstick blood test has been developed by Cepheid (the Xpert MTB Host Response [MTB-HR] prototype), which generates a "TB score" based on messenger RNA (mRNA) expression of 3 genes. Here we describe the first prospective findings of the MTB-HR prototype., Methods: Fingerstick blood from adults presenting with symptoms compatible with TB in South Africa, The Gambia, Uganda, and Vietnam was analyzed using the Cepheid GeneXpert MTB-HR prototype. Accuracy of the Xpert MTB-HR cartridge was determined in relation to GeneXpert Ultra results and a composite microbiological score (GeneXpert Ultra and liquid culture) with patients classified as having TB or other respiratory diseases (ORD)., Results: When data from all sites (n = 75 TB, 120 ORD) were analyzed, the TB score discriminated between TB and ORD with an area under the curve (AUC) of 0.94 (95% confidence interval [CI], .91-.97), sensitivity of 87% (95% CI, 77-93%) and specificity of 94% (88-97%). When sensitivity was set at 90% for a triage test, specificity was 86% (95% CI, 75-97%). These results were not influenced by human immunodeficiency virus (HIV) status or geographical location. When evaluated against a composite microbiological score (n = 80 TB, 111 ORD), the TB score was able to discriminate between TB and ORD with an AUC of 0.88 (95% CI, .83-.94), 80% sensitivity (95% CI, 76-85%) and 94% specificity (95% CI, 91-96%)., Conclusions: Our interim data indicate the Cepheid MTB-HR cartridge reaches the minimal target product profile for a point of care triage test for TB using fingerstick blood, regardless of geographic area or HIV infection status., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Objective: Hemarthrosis after knee trauma often indicates serious joint injury. Few studies have evaluated agreement between clinical examination and findings from magnetic resonance imaging (MRI). We aimed to describe the agreement between acute clinical examination and subacute MRI findings after acute knee trauma with hemarthrosis and the importance of the subspecialty of the examiner., Design: Longitudinal cohort study. Agreement with MRI findings was evaluated by logistic regression., Setting: Helsingborg hospital., Patients: Thousand one hundred forty-five consecutive patients with hemarthrosis after knee trauma., Interventions: Clinical examination and MRI., Main Outcome Measures: agreement between clinical examination and findings from MRI. We considered the radiologist's report as the gold standard., Results: Median time (25th, 75th percentile) from injury to clinical examination was 2 (1, 7) days, and from injury to imaging was 8 (5, 15) days. The overall sensitivity and specificity of clinical examination versus MRI for major ligament injury or lateral patella dislocation (LPD) were 70% [95% confidence interval 67-73) and 66% (61-72), respectively. Orthopedic subspecialist knee had the highest agreement with anterior cruciate ligament rupture (adjusted odds ratios were 1.7 (95% confidence interval 1.2-2.3), 1.9 (1.2-3.0) and 5.9 (3.7-9.5) for orthopedic trainees, orthopedic subspecialists other, and orthopedic subspecialist knee, respectively]. For other ligament injuries and LPD, we did not find statistically significant differences., Conclusions: Clinical diagnosis after acute knee injury is relatively unreliable versus MRI findings even when performed by orthopedic specialists. However, the agreement is improved when the examination is performed by an orthopedic knee subspecialist., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Despite microbiological cure, about 50% of tuberculosis (TB) patients have poor lung recovery. Neutrophils are associated with lung pathology; however, CD16/CD62L-defined subsets have not been studied in TB. Using flow cytometry, we monitored frequencies, phenotype, and function of neutrophils following stimulation with Mycobacterium tuberculosis (Mtb) whole cell lysate (WCL) and ESAT-6/CFP-10 fusion protein (EC) in relation to lung pathology., Methods: Fresh blood from 42 adult, human immunodeficiency virus (HIV)-negative TB patients were analyzed pre- and post-therapy, with disease severity determined using chest radiography and bacterial load. Flow cytometry was used to monitor frequencies, phenotype, and function (generation of reactive oxygen species [ROS], together with CD11b, tumor necrosis factor, and interleukin 10 [IL-10] expression) of neutrophils following 2-hour stimulation with Mtb-specific antigens., Results: Total neutrophils decreased by post-treatment compared to baseline (P = .0059); however, CD16brCD62Lbr (segmented) neutrophils increased (P = .0031) and CD16dimCD62Lbr (banded) neutrophils decreased (P = .038). Banded neutrophils were lower in patients with severe lung damage at baseline (P = .035). Following WCL stimulation, ROS from segmented neutrophils was higher in patients with low Mtb loads even after adjusting for sex (P = .038), whereas IL-10-expressing CD16dimCD62Llo cells were higher in patients with mild damage (P = .0397) at baseline., Conclusions: High ROS generation, low levels of banded neutrophils, and high levels of IL-10-expressing CD16dimCD62Llo neutrophils are associated with reduced lung pathology at diagnosis. Hence, neutrophils are potential early indicators of TB severity and promising targets for TB host-directed therapy., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Background: The inflammatory response to Mycobacterium tuberculosis results in variable degrees of lung pathology during active TB (ATB) with central involvement of neutrophils. Little is known about neutrophil-derived mediators and their role in disease severity at baseline and recovery upon TB treatment initiation., Methods: 107 adults with confirmed pulmonary TB were categorised based on lung pathology at baseline and following successful therapy using chest X-ray scores (Ralph scores) and GeneXpert bacterial load (Ct values). Plasma, sputum, and antigen-stimulated levels of MMP1, MMP3, MMP8, MMP9, MPO, S100A8/9, IL8, IL10, IL12/23(p40), GM-CSF, IFNγ, and TNF were analysed using multiplex cytokine arrays., Results: At baseline, neutrophil counts correlated with plasma levels of MMP8 (rho = 0.45, p = 2.80E-06), S100A8 (rho = 0.52, p = 3.00E-08) and GM-CSF (rho = 0.43, p = 7.90E-06). Levels of MMP8 (p = 3.00E-03), MMP1 (p = 1.40E-02), S100A8 (p = 1.80E-02) and IL12/23(p40) (p = 1.00E-02) were associated with severe lung damage, while sputum MPO levels were directly linked to lung damage (p = 1.80E-03), Mtb load (p = 2.10E-02) and lung recovery (p = 2.40E-02). Six months of TB therapy significantly decreased levels of major neutrophil-derived pro-inflammatory mediators: MMP1 (p = 4.90E-12 and p = 2.20E-07), MMP8 (p = 3.40E-14 and p = 1.30E-05) and MMP9 (p = 1.60E-04 and p = 1.50E-03) in plasma and sputum, respectively. Interestingly, following H37Rv whole cell lysate stimulation, S100A8 (p = 2.80E-02), MMP9 (p = 3.60E-02) and MPO (p = 9.10E-03) levels at month 6 were significantly higher compared to baseline. Sputum MMP1 (p = 1.50E-03), MMP3 (p = 7.58E-04), MMP9 (p = 2.60E-02) and TNF (p = 3.80E-02) levels were lower at month 6 compared to baseline in patients with good lung recovery., Conclusion: In this study, patients with severe lung pathology at baseline and persistent lung damage after treatment were associated with higher plasma and sputum levels of major pro-inflammatory neutrophil-derived mediators. Interestingly, low sputum MPO levels were associated with severe lung damage, higher Mtb burden and low recovery. Our data suggest that therapeutic agents which target these mediators should be considered for future studies on biomarkers and host-directed therapeutic approaches against TB-related lung pathology and/or lung recovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Muefong, Owolabi, Donkor, Charalambous, Mendy, Sey, Bakuli, Rachow, Geldmacher and Sutherland.)

The development of a non-sputum-based, point-of-care diagnostic test for tuberculosis (TB) is a priority in the global effort to combat this disease, particularly in resource-constrained settings. Previous studies have identified host biomarker signatures which showed potential, but there is a need to validate and refine these for development as a test. We recruited 1,403 adults presenting with symptoms suggestive of pulmonary TB at primary healthcare clinics in six countries from West, East and Southern Africa. Of the study cohort, 326 were diagnosed with TB and 787 with other respiratory diseases, from whom we randomly selected 1005 participants. Using Luminex ® technology, we measured the levels of 20 host biomarkers in serum samples which we used to evaluate the diagnostic accuracy of previously identified and novel bio-signatures. Our previously identified seven-marker bio-signature did not perform well (sensitivity: 89%, specificity: 60%). We also identified an optimal, two-marker bio-signature with a sensitivity of 94% and specificity of 69% in patients with no history of previous TB. This signature performed slightly better than C-reactive protein (CRP) alone. The cut-off value for a positive diagnosis differed for human immuno-deficiency virus (HIV)-positive and -negative individuals. Notably, we also found that no signature was able to diagnose TB adequately in patients with a prior history of the disease. We have identified a two-marker, pan-African bio-signature which is more robust than CRP alone and meets the World Health Organization (WHO) target product profile requirements for a triage test in both HIV-negative and HIV-positive individuals. This signature could be incorporated into a point-of-care device, greatly reducing the necessity for expensive confirmatory diagnostics and potentially reducing the number of cases currently lost to follow-up. It might also potentially be useful with individuals unable to provide sputum or with paucibacillary disease. We suggest that the performance of TB diagnostic signatures can be improved by incorporating the HIV-status of the patient. We further suggest that only patients who have never had TB be subjected to a triage test and that those with a history of previous TB be evaluated using more direct diagnostic techniques., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mutavhatsindi, van der Spuy, Malherbe, Sutherland, Geluk, Mayanja-Kizza, Crampin, Kassa, Howe, Mihret, Sheehama, Nepolo, Günther, Dockrell, Corstjens, Stanley, Walzl, Chegou and the AE-TBC ScreenTB Consortia.)

In Indonesia, BCG vaccine protection against Mycobacterium tuberculosis infection decreased with increasing exposure to the pathogen. We aimed to validate these findings in Africa. Poisson regression was used to estimate BCG protection, stratified by pathogen exposure using an exposure score, against enzyme-linked immunospot assay conversion at 3 months in 220 Gambian case contacts. Although the interaction between BCG and exposure was not significant (P = .13), BCG protection was strongest in the lowest-exposure tertile (relative risk, 0.35 [95% confidence interval, .15-.82; P = .02] vs 0.50 [.30-.83; P = .008] and 0.71 (.45-1.13; P = .1] for the middle and highest-exposure tertiles, respectively. These results are consistent with those from Indonesia., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Blood-based biomarkers have been proposed as an alternative to current sputum-based treatment monitoring methods in active tuberculosis (ATB). The aim of this study was to validate previously described phenotypic, activation, and cytokine markers of treatment response in a West African cohort., Methods: Whole blood immune responses to Mycobacterium tuberculosis ESAT-6/CFP-10 (EC) and purified protein derivative (PPD) were measured in twenty adults at baseline and after 2 months of standard TB treatment. Patients were classified as fast or slow responders based on a negative or positive sputum culture result at 2 months, respectively. Cellular expression of activation markers (CD38, HLA-DR), memory markers (CD27), and functional intracellular cytokine and proliferation (IFN-γ, Ki-67, TNF-α) markers were measured using multi-color flow cytometry., Results: There was a significant increase in the proportion of CD4 + CD27 + cells expressing CD38 and HLA-DR following EC stimulation at 2 months compared to baseline ( p = 0.0328 and p = 0.0400, respectively). Following PPD stimulation, slow treatment responders had a significantly higher proportion of CD8 + CD27 - IFN-γ + ( p = 0.0105) and CD4 + CD27 + HLA-DR + CD38 + ( p = 0.0077) T cells than fast responders at baseline. Receiver operating curve analysis of these subsets resulted in 80% sensitivity and 70 and 100% specificity, respectively (AUC of 0.82, p = 0.0156 and 0.84, p = 0.0102)., Conclusion: Our pilot data show reductions in expression of T cell activation markers were seen with treatment, but this was not associated with fast or slow sputum conversion at 2 months. However, baseline proportions of activated T cell subsets are potentially predictive of the subsequent speed of response to treatment., (Copyright © 2020 Vickers, Darboe, Muefong, Mbayo, Barry, Gindeh, Njie, Riley, Sarr, Sambou, Dockrell, Charalambous, Rachow, Owolabi, Jayasooriya and Sutherland.)

Background: While T cell responses to Mycobacterium tuberculosis (Mtb) have been extensively studied, the role of B-cells and antibodies are less well characterised. The aim of this study was to assess levels of Mtb-specific IgG + plasmablasts across the Mtb infection spectrum., Methods: Patients with active TB were analysed at baseline and 6 months of therapy (n = 20).Their exposed household contacts (HHC) included individuals with latent TB infection (LTBI; n = 20); evident at baseline; individuals with a negative Tuberculin Skin Test (TST) at baseline who became; positive at 6 months (converters; n = 11) and those who remained negative (non-converters; n = 10). An e x-vivo B-cell ELISPOT was performed to analyse plasmablast responses., Results: Frequencies of ESAT-6/CFP-10 (EC)- but not Whole Cell Lysate (WCL)-specific plasmablasts were significantly higher in patients with active TB pre-treatment compared to post-treatment (p = 0.002) and compared to HHC with LTBI (p < 0.0001). Conversely, total IgG + plasmablasts were significantly decreased in TB patients at baseline. No difference was seen in levels of plasmablasts between TST converters and non-converters at baseline., Conclusions: We show that EC-specific plasmablast levels are differentially modulated during TB infection and disease, with levels highest during active TB. These data provide new insights into TB biomarker development and avenues for novel immune interventions., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Interferon-γ release assays (IGRA) with Resuscitation promoting factor (Rpf) proteins enhanced tuberculosis (TB) screening and diagnosis in adults but have not been evaluated in children. Children often develop paucibacillary TB and their immune response differs from that of adults, which together affect TB disease diagnostics and immunodiagnostics. We assessed the ability of Rpf to identify infection among household TB-exposed children in The Gambia and investigated their ability to discriminate Mycobacterium tuberculosis complex (MTBC) infection from active TB disease in children., Methods: Detailed clinical investigations were done on 93 household TB-exposed Gambian children and a tuberculin skin test (TST) was administered to asymptomatic children. Venous blood was collected for overnight stimulation with ESAT-6/CFP-10-fusion protein (EC), purified protein derivative and RpfA, B, C, D and E. Interferon gamma (IFN-γ) production was measured by ELISA in supernatants and corrected for the background level. Infection status was defined by IGRA with EC and TB disease by mycobacterial confirmation and/or clinical diagnosis. We compared IFN-γ levels between infected and uninfected children and between infected and TB diseased children using a binomial logistic regression model while correcting for age and sex. A Receiver Operating Characteristics analysis was done to find the best cut-off for IFN-γ level and calculate sensitivity and specificity., Results: Interferon gamma production was significantly higher in infected (IGRA+, n = 45) than in uninfected (IGRA-, n = 20) children after stimulation with RpfA, B, C, and D (P = 0.03; 0.007; 0.03 and 0.003, respectively). Using RpfB and D-specific IFN-γ cut-offs (33.9 pg/mL and 67.0 pg/mL), infection was classified with a sensitivity-specificity combination of 73-92% and 77-72% respectively, which was similar to and better than 65-75% for TST. Moreover, IFN-γ production was higher in infected than in TB diseased children (n = 28, 5 bacteriologically confirmed, 23 clinically diagnosed), following RpfB and D stimulation (P = 0.02 and 0.03, respectively)., Conclusion: RpfB and RpfD show promising results for childhood MTBC infection screening, and both performed similar to and better than the TST in our study population. Additionally, both antigens appear to discriminate between infection and disease in children and thus warrant further investigation as screening and diagnostic antigens for childhood TB.

Background: Strategies to prevent Mycobacterium tuberculosis (Mtb) infection are urgently required. In this study, we aimed to identify correlates of protection against Mtb infection., Methods: Two groups of Mtb-exposed contacts of tuberculosis (TB) patients were recruited and classified according to their Mtb infection status using the tuberculin skin test (TST; cohort 1) or QuantiFERON (QFT; cohort 2). A negative reading at baseline with a positive reading at follow-up classified TST or QFT converters and a negative reading at both time points classified TST or QFT nonconverters. Ribonucleic acid sequencing, Mtb proteome arrays, and metabolic profiling were performed., Results: Several genes were found to be differentially expressed at baseline between converters and nonconverters. Gene set enrichment analysis revealed a distinct B-cell gene signature in TST nonconverters compared to converters. When infection status was defined by QFT, enrichment of type I interferon was observed. A remarkable area under the curve (AUC) of 1.0 was observed for IgA reactivity to Rv0134 and an AUC of 0.98 for IgA reactivity to both Rv0629c and Rv2188c. IgG reactivity to Rv3223c resulted in an AUC of 0.96 and was markedly higher compared to TST nonconverters. We also identified several differences in metabolite profiles, including changes in biomarkers of inflammation, fatty acid metabolism, and bile acids. Pantothenate (vitamin B5) was significantly increased in TST nonconverters compared to converters at baseline (q = 0.0060)., Conclusions: These data provide new insights into the early protective response to Mtb infection and possible avenues to interfere with Mtb infection, including vitamin B5 supplementation.Analysis of blood from highly exposed household contacts from The Gambia who never develop latent Mycobacterium tuberculosis infection shows distinct transcriptomic, antibody, and metabolomic profiles compared to those who develop latent tuberculosis infection but prior to any signs of infection., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Protective immunity to Mycobacterium tuberculosis (Mtb) -the causative agent of tuberculosis (TB)-is not fully understood but involves immune responses within the pulmonary airways which can lead to exacerbated inflammation and immune pathology. In humans, this inflammation results in lung damage; the extent of which depends on specific host pro-inflammatory processes. Neutrophils, though increasingly linked to the development of inflammatory disorders, have been less well studied in relation to TB-induced lung pathology. Neutrophils mode of action and their specialized functions can be directly linked to TB-specific lung tissue damage observed on patient chest X-rays at diagnosis and contribute to long-term pulmonary sequelae. This review discusses aspects of neutrophil activity associated with active TB, including the resulting inflammation and pulmonary impairment. It highlights the significance of neutrophil function on TB disease outcome and underlines the necessity of monitoring neutrophil function for better assessment of the immune response and severity of lung pathology associated with TB. Finally, we propose that some MMPs, ROS, MPO, S100A8/A9 and Glutathione are neutrophil-related inflammatory mediators with promising potential as targets for developing host-directed therapies for TB., (Copyright © 2020 Muefong and Sutherland.)

Background: To facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify novel candidate host biomarkers. SERPING1, which encodes C1-inhibitor (C1-INH), the natural inhibitor of the C1-complex has emerged as candidate biomarker. Here we collated and analysed SERPING1 expression data and subsequently determined C1-INH protein levels in four cohorts of patients with TB., Methods: SERPING1 expression data were extracted from online deposited datasets. C1-INH protein levels were determined by ELISA in sera from individuals with active TB, LTBI as well as other disease controls in geographically diverse cohorts., Findings: SERPING1 expression was increased in patients with active TB compared to healthy controls (8/11 cohorts), LTBI (13/14 cohorts) and patients with other (non-TB) lung-diseases (7/7 cohorts). Serum levels of C1-INH were significantly increased in The Gambia and Italy in patients with active TB relative to the endemic controls but not in South Africa or Korea. In the largest cohort (n = 50), with samples collected longitudinally, normalization of C1-INH levels following successful TB treatment was observed. This cohort, also showed the most abundant increase in C1-INH, and a positive correlation between C1q and C1-INH levels. Combined presence of increased levels of both C1q and C1-INH had high specificity for active TB (96 %) but only very modest sensitivity 38 % compared to the endemic controls., Interpretation: SERPING1 transcript expression is increased in TB patients, while serum protein levels of C1-INH were increased in half of the cohorts analysed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We perform single-shot frequency domain holography to measure the ultrafast spatio-temporal phase change induced by the optical Kerr effect and plasma in flexible Corning Willow Glass during femtosecond laser-matter interactions. We measure the nonlinear index of refraction ($ {n&#95;2} $n 2 ) to be $(3.6 \pm 0.1) \times {10^{ - 16}}\;{{\rm cm}^2}/{\rm W} $(3.6±0.1)×10 -16 cm 2 /W and visualize the plasma formation and recombination on femtosecond time scales in a single shot. To compare with the experiment, we carry out numerical simulations by solving the nonlinear envelope equation.

Background: Tuberculosis (TB) is the leading cause of death from a single infectious agent worldwide. The immune system is capable of clearing the pathogen before establishment of latent infection but the mechanisms for this are not yet understood., Methods: This study analysed highly exposed household contacts (HHC) of TB index cases who were categorised according to QuantiFERON (QFT) results at recruitment and 6 months. Seventeen (17) QFT nonconverters, 14 QFT converters, 18 QFT reverters and 18 latent TB infection (LTBI) were analysed. Supernatants generated following QFT stimulation at both time-points were analysed using a 64-plex cytokine array. Flow cytometry was performed on QFT converters and nonconverters at baseline only., Results: Interleukin-2 (IL-2), IL-5, IL-13, APRIL, IL-17A, IP-10, MIP-1ß, sIL-6rb, OPN, and sTNFR2 were all significantly higher in the QFT converters compared with nonconverters at baseline. Levels of interferon-α2 (IFN-α2) and IL-2 were significantly lower in QFT reverters compared with nonconverters at baseline. Analysis of Ag-specific IL-2 levels resulted in an area under the curve (AUC) of 0.93 (95% confidence interval [CI], 0.84-1.00) for QFT converters compared to nonconverters and an AUC of 0.80 (0.65-0.95) for QFT reverters compared with LTBI. Purified protein derivative (PPD)-specific CD4 + CD26 + IFN-γ + cells were significantly increased (P = .0007) in QFT nonconverters compared with QFT converters at baseline., Conclusions: Our results provide insight into the underlying mechanisms of resistance to sustained Mycobacterium tuberculosis infection., (© 2019 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

There remains a pressing need for biomarkers that can predict who will progress to active tuberculosis (TB) after exposure to Mycobacterium tuberculosis (MTB) bacterium. By analyzing cohorts of household contacts of TB index cases (HHCs) and a stringent non-human primate (NHP) challenge model, we evaluated whether integration of blood transcriptional profiling with serum metabolomic profiling can provide new understanding of disease processes and enable improved prediction of TB progression. Compared to either alone, the combined application of pre-existing transcriptome- and metabolome-based signatures more accurately predicted TB progression in the HHC cohorts and more accurately predicted disease severity in the NHPs. Pathway and data-driven correlation analyses of the integrated transcriptional and metabolomic datasets further identified novel immunometabolomic signatures significantly associated with TB progression in HHCs and NHPs, implicating cortisol, tryptophan, glutathione, and tRNA acylation networks. These results demonstrate the power of multi-omics analysis to provide new insights into complex disease processes.

Background: Prior Influenza A viral (IAV) infection has been shown to increase susceptibility to tuberculosis (TB) and TB has also been shown to be a primary cause of death during pandemics, including the Spanish Influenza outbreak of 1918-1919. The majority of data has been obtained from mouse models, thus the aim of this study was to determine the impact of Flu co-infection on host immunity and disease severity in TB patients at diagnosis. Methods: Sputum from 282 patients with active TB were analyzed for presence of FluA/FluB RNA at presentation using multiplex PCR. Sputum RNA was also analyzed for Mycobacterium tuberculosis (Mtb) load using 16 S RNA amplification. Supernatants from digested sputum and Mtb antigen-stimulated whole blood were analyzed using multiplex cytokine arrays and PBMC were analyzed for cytokine production from CD4+ T, CD8+ T and Mucosal Associated Invariant T cells (MAITs). Results: 12 (4.3%) of TB patients were found to have FluA or FluB viral RNA present in their sputum at the time of TB diagnosis. The TB/Flu co-infected patients had a significantly higher bacterial load compared to those with TB mono-infection ( p = 0.0026). They had lower levels of IL17A in ex vivo sputum ( p = 0.0275) and higher MCP-1 (CCL2) levels in the blood following PPD stimulation ( p = 0.0267). TB/Flu co-infected subjects had significantly higher IFN-γ+IL-17+CD4+ and IFN-γ+IL-17-CD8+ cells compared to TB mono-infected subjects. Conclusions: These data show that Flu co-infection at time of TB diagnosis is associated with a higher bacterial load and differential cellular and soluble profiles. These findings show for the first time the impact of TB/Flu co-infection in a human cohort and support the potential benefit of Flu vaccination in TB-endemic settings.

New biomarkers of tuberculosis (TB) risk and disease are critical for the urgently needed control of the ongoing TB pandemic. In a prospective multisite study across Subsaharan Africa, we analyzed metabolic profiles in serum and plasma from HIV-negative, TB-exposed individuals who either progressed to TB 3-24 months post-exposure (progressors) or remained healthy (controls). We generated a trans-African metabolic biosignature for TB, which identifies future progressors both on blinded test samples and in external data sets and shows a performance of 69% sensitivity at 75% specificity in samples within 5 months of diagnosis. These prognostic metabolic signatures are consistent with development of subclinical disease prior to manifestation of active TB. Metabolic changes associated with pre-symptomatic disease are observed as early as 12 months prior to TB diagnosis, thus enabling timely interventions to prevent disease progression and transmission.

Background: Tuberculosis (TB) remains a major threat to global health. Currently, diagnosis of active TB is hampered by the lack of specific biomarkers that discriminate active TB disease from other (lung) diseases or latent TB infection (LTBI). Integrated human gene expression results have shown that genes encoding complement components, in particular different C1q chains, were expressed at higher levels in active TB compared to LTBI. Methods: C1q protein levels were determined using ELISA in sera from patients, from geographically distinct populations, with active TB, LTBI as well as disease controls. Results: Serum levels of C1q were increased in active TB compared to LTBI in four independent cohorts with an AUC of 0.77 [0.70; 0.83]. After 6 months of TB treatment, levels of C1q were similar to those of endemic controls, indicating an association with disease rather than individual genetic predisposition. Importantly, C1q levels in sera of TB patients were significantly higher as compared to patients with sarcoidosis or pneumonia, clinically important differential diagnoses. Moreover, exposure to other mycobacteria, such as Mycobacterium leprae (leprosy patients) or BCG (vaccinees) did not result in elevated levels of serum C1q. In agreement with the human data, in non-human primates challenged with Mycobacterium tuberculosis , increased serum C1q levels were detected in animals that developed progressive disease, not in those that controlled the infection. Conclusions: In summary, C1q levels are elevated in patients with active TB compared to LTBI in four independent cohorts. Furthermore, C1q levels from patients with TB were also elevated compared to patients with sarcoidosis, leprosy and pneumonia. Additionally, also in NHP we observed increased C1q levels in animals with active progressive TB, both in serum and in broncho-alveolar lavage. Therefore, we propose that the addition of C1q to current biomarker panels may provide added value in the diagnosis of active TB.

Tuberculosis (TB) is still a global health concern, especially in resource-poor countries such as The Gambia. Defining protective immunity to TB is challenging: its pathogenesis is complex and involves several cellular components of the immune system. Recent works in vaccine development suggest important roles of the innate immunity in natural protection to TB, including natural killer (NK) cells. NK cells mediate cellular cytotoxicity and cytokine signaling in response to Mycobacterium tuberculosis (Mtb). NK cells can display specific memory-type markers to previous antigen exposure; thus, bridging innate and adaptive immunity. However, major knowledge gaps exist on the contribution of NK cells in protection against Mtb infection or TB. We performed a cross-sectional assessment of NK cells phenotype and function in four distinct groups of individuals: TB cases pre-treatment ( n  = 20) and post-treatment ( n  = 19), and household contacts with positive ( n  = 9) or negative ( n  = 18) tuberculin skin test (TST). While NK cells frequencies were similar between all groups, significant decreases in interferon-γ expression and degranulation were observed in NK cells from TB cases pre-treatment compared to post-treatment. Conversely, CD57 expression, a marker of advanced NK cells differentiation, was significantly lower in cases post-treatment compared to pre-treatment. Finally, NKG2C, an activation and imprinted-NK memory marker, was significantly increased in TST+ (latently infected) compared to TB cases pre-treatment and TST- (uninfected) individuals. The results of this study provide valuable insights into the role of NK cells in Mtb infection and TB disease, demonstrating potential markers for distinguishing between infection states and monitoring of TB treatment response.

We investigated host-derived biomarkers that were previously identified in QuantiFERON supernatants, in a large pan-African study. We recruited individuals presenting with symptoms of pulmonary TB at seven peripheral healthcare facilities in six African countries, prior to assessment for TB disease. We then evaluated the concentrations of 12 biomarkers in stored QuantiFERON supernatants using the Luminex platform. Based on laboratory, clinical and radiological findings and a pre-established algorithm, participants were classified as TB disease or other respiratory diseases(ORD). Of the 514 individuals included in the study, 179(34.8%) had TB disease, 274(51.5%) had ORD and 61(11.5%) had an uncertain diagnosis. A biosignature comprising unstimulated IFN-γ, MIP-1β, TGF-α and antigen-specific levels of TGF-α and VEGF, identified on a training sample set (n = 311), validated by diagnosing TB disease in the test set (n = 134) with an AUC of 0.81(95% CI, 0.76-0.86), corresponding to a sensitivity of 64.2%(95% CI, 49.7-76.5%) and specificity of 82.7%(95% CI, 72.4-89.9%). Host biomarkers detected in QuantiFERON supernatants can contribute to the diagnosis of active TB disease amongst people presenting with symptoms requiring investigation for TB disease, regardless of HIV status or ethnicity in Africa.

IL-17-producing cells have been shown to be important in the early stages of Mycobacterium tuberculosis (Mtb) infection in animal models. However, there are very little data on the role of IL-17 in human studies of tuberculosis (TB). We recruited TB patients and their highly exposed contacts who were further categorized based on results from an IFN-γ-release assay (IGRA): (1) IGRA positive (IGRA + ) at recruitment (latently TB infected), (2) IGRA negative (IGRA - ) at recruitment and 6 months [non-converters (NC)], and (3) IGRA - at recruitment and IGRA + at 6 months (converters). Whole blood was stimulated with mycobacterial antigens and analyzed using T helper (Th) 17 multiplex cytokine assays. Th17, Vγ9Vδ2 + , and CD161 ++ Vα7.2 + mucosal-associated invariant T (MAIT) cells were analyzed by flow cytometry. The majority of IL-17 was produced by CD26 + CD4 + Th17 cells (median 71%) followed by γδ T cells (6.4%) and MAIT cells (5.8%). TB patients had a significantly lower proportion of Th17 cells and CD4 + CD161 + Vα7.2 + cells producing both IL-17 and IFN-γ compared to LTBI subjects. IGRA NC had significantly lower levels of CD26 - CD4 + and CD8 + MAIT cells producing IL-17 compared to IGRA C but had significantly higher levels of IL-17A, IL-17F, IL-21, and IL-23 in ESAT-6/CFP-10-stimulated supernatants compared to IGRA C. These data provide new insights into the role of IL-17 and IL-17-producing cells at three key stages of the Mtb infection spectrum.

Introduction: The currently used Streptococcus pneumoniae vaccines have had a significant impact on the pneumococcal diseases caused by the serotypes they cover. Their limitations have stimulated a search for alternate vaccines that will cover all serotypes, be affordable and effective in young children. Pneumococcal protein antigens are potential vaccine candidates that may meet some of the shortfalls of the current vaccines. Thus, this study aimed to determine the relationship between antibodies against pneumococcal protein antigens and nasopharyngeal carriage in infants., Methods: One hundred and twenty mother-infant pairs were enrolled into the study. They had nasopharyngeal swabs(NPS) taken at birth and every two weeks for the first eight weeks after delivery, and blood samples were obtained at birth and every four weeks for the first eight weeks after delivery. Nasopharyngeal carriage of S. pneumoniae was determined from the NPS and antibodies against the pneumococcal proteins CbpA, PspA and rPly were measured in the blood samples., Results: The S. pneumoniae carriage rate in infants increased to that of mothers by eight weeks of age. The odds of carriage in infants was 6.2 times (95% CI: 2.0-18.9) higher when their mothers were also carriers. Bacterial density in infants was lower at birth compared to their mothers (p = 0.004), but increased with age and became higher than that of their mothers at weeks 4 (p = 0.009), 6 (p = 0.002) and 8 (p<0.0001). At birth, the infants' antibodies against CbpA, and rPly pneumococcal protein antigens were similar, but that of PspA was lower (p<0.0001), compared to their mothers. Higher antibody concentrations to CbpA [OR (95% CI): 0.49 (0.26-0.92, p = 0.03)], but not PspA and rPly, were associated with protection against carriage in the infants., Conclusion: Naturally induced antibodies against the three pneumococcal protein antigens were transferred from mother to child. The proportion of infants with nasopharyngeal carriage and the bacterial density of S. pneumoniae increased with age within the first eight weeks of life. Higher concentrations of antibodies against CbpA, but not PspA and rPly, were associated with reduced risk of nasopharyngeal carriage of S. pneumoniae in infants.

Bacillus Calmette-Guérin (BCG), the only licensed vaccine against tuberculosis, has been shown to provide heterologous protection against unrelated pathogens and enhance antibody responses to several routine expanded program on immunization (EPI) vaccines. Understanding these heterologous effects is important for the development of optimal vaccination strategies. We set out to assess the effect of vaccination with BCG Russia of 6-week-old infants on in vitro reactivity to a panel of toll-like receptor (TLR) agonists (TLR2, 4, and 7/8) and heat-killed pathogens [ Streptococcus pneumoniae, Candida albicans (CA), and Escherichia coli ], and antibody responses to other EPI vaccines compared to BCG naïve infants. We observed no effect of BCG vaccination on innate (TNF-α) or Th2 (IL-4) cytokine responses, but found enhanced CA-specific CD8 + IFN-γ + responses in BCG vaccinated males and females 1 week after vaccination and decreased IFN-γ:IL4 ratio to SP in females. By 12 weeks (but not 1 week) of post-vaccination, there was significant downmodulation of Th1 cytokine responses in BCG vaccinated infants; and TLR-stimulated IL-10 and IL-17 responses declined in BCG vaccinated females but not males. Significant changes also occurred in the BCG naïve group, mainly at 18 weeks, including decreased Th1 and increased IL-10 responses. The effects at 18 weeks were most likely a result of immune modulation by the intervening EPI vaccines given at 8, 12, and 16 weeks of age. There was no effect of BCG vaccination on EPI antibody levels at either time point. Taken together, our results support minimal early heterologous immune modulation by BCG Russia vaccination that did not persist 12 weeks after vaccination.

Background: Tuberculosis (TB) has overtaken HIV as the biggest infectious disease killer, with the majority of deaths occurring in sub-Saharan Africa. However it is unknown how differences in bacterial load alter host immune profiles in the sputum and blood of TB patients., Methods: 16S ribosomal RNA analysis was used to determine bacterial load in sputum samples obtained from 173 patients with active TB (57 pre-treatment and 116 post-treatment). Host analyte concentrations in sputum and Mycobacterium tuberculosis (Mtb) antigen stimulated whole blood assay supernatants were analysed using multiplex cytokine arrays., Results: Multiple logistic regression adjusting for age, sex and HIV status showed highly significant correlation of bacterial load with IL1β, IL2, IL1RA, IL4, IL6, IL8, IL9, IL15, IL17, EOTAX, FGF, IFN-γ, GCSF, MCP1, M1P1α, M1P1β, PDGF, TNFα, VEGF in sputum. With increasing time on treatment, FGF levels in sputum displayed the most significant inverse correlation with reduction in bacterial load., Conclusions: We show that differences in bacterial load correlates with changes in several host biomarkers. These findings have implications for development of tests for TB diagnosis and treatment response., Competing Interests: The authors have declared that no competing interests exist.

Objectives/background: Mycobacterium africanum that causes 40% of tuberculosis (TB) in West Africa grows more slowly in culture and has similar transmission capacity compared with Mycobacterium tuberculosis, but M. africanum-exposed contacts progress more slowly to active disease. The presence of lipid body (LB) containing M. tuberculosis complex (MTBC) cells in sputum samples has been associated with mycobacterial transcriptomes indicating slow or no growth and persister-like antibiotic tolerance. Slow-growing bacilli have been found to display a persister-like phenotype with the accumulation of LBs and drug tolerance. Our previous study showed that the body mass index and lung damage resolution on chest X-ray were significantly improved slower in M. africanum-infected patients posttreatment than in M. tuberculosis-infected patients; however, the reason for this remains unclear. Therefore, we hypothesized that these differences could be either due to significant differences in drug resistance between the MTBC lineages or a difference in their content of persisters, as indicated by the percentage of LP-positive bacilli in sputum., Methods: Sputum isolates collected before treatment from patients with TB were subjected to drug susceptibility testing using the BD BACTEC MGIT 960 SIRE kit. The percentage of acid-fast bacilli (AFB) and LB-positive bacilli in pretreatment sputum was determined by a dual staining procedure using Auramine O and LipidTOX Red neutral lipid stain, respectively, and fluorescence microscopy imaging., Results: Out of the 77 isolates tested, 9 showed resistance to at least one drug and only 2 showed multidrug (rifampicin and isoniazid) resistance among M. tuberculosis-infected patients. The percentage of AFB-positive smears was similar between the two groups (p=0.821), whereas that of LP-positive bacilli was significantly higher (p=0.0059) in M. africanum-infected patients' sputa (n=24) than in M. tuberculosis-infected patients' sputa (n=36). In addition, the bacillary lengths were significantly higher in M. africanum-infected patients' sputa than in M. tuberculosis-infected patients' sputa (p=0.0007). A high frequency of LP-positive bacilli in pretreatment sputum was associated with a poor body mass index and lung damage on chest X-ray improvement following anti-TB treatment in both the groups (r 2 =0.022; p=0.017)., Conclusion: The slow clinical recovery of M. africanum-infected patients compared with M. tuberculosis-infected patients posttreatment may be at least partially associated with the persistence of drug-tolerant "fat and lazy" bacilli., (Copyright © 2016.)

Vaccines can have nontargeted heterologous effects that manifest as increased protection against nonvaccine infections, as described for measles vaccine (MV), or increased susceptibility to infections and death, as described following diphtheria-tetanus-whole cell pertussis (DTP) vaccination. The mechanisms are unknown, and high-quality immunological studies are lacking. This study was designed to investigate the heterologous effects of MV and DTP in 302 Gambian infants. The results support a sex-differential immunosuppressive effect of DTP on innate proinflammatory responses and T-cell immunity. Males but not females receiving MV had enhanced proinflammatory innate responses. The results point to modified signaling via Toll-like receptor 4 (TLR4) as a possible mechanism for the effects on innate immunity. When both vaccines were administered together, purified protein derivative responses were enhanced in females but downregulated in males. Collectively, these data indicate immunological effects that could account for heterologous effects of MV and DTP, to take forward into prospective trials., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

High risk human papilloma viruses (HPVs) may have a causal role in some breast cancers. Case-control studies, conducted in many different countries, consistently indicate that HPVs are more frequently present in breast cancers as compared to benign breast and normal breast controls (odds ratio 4.02). The assessment of causality of HPVs in breast cancer is difficult because (i) the HPV viral load is extremely low, (ii) HPV infections are common but HPV associated breast cancers are uncommon, and (iii) HPV infections may precede the development of breast and other cancers by years or even decades. Further, HPV oncogenesis can be indirect. Despite these difficulties, the emergence of new evidence has made the assessment of HPV causality, in breast cancer, a practical proposition. With one exception, the evidence meets all the conventional criteria for a causal role of HPVs in breast cancer. The exception is "specificity." HPVs are ubiquitous, which is the exact opposite of specificity. An additional reservation is that the prevalence of breast cancer is not increased in immunocompromised patients as is the case with respect to HPV-associated cervical cancer. This indicates that HPVs may have an indirect causal influence in breast cancer. Based on the overall evidence, high-risk HPVs may have a causal role in some breast cancers.

Objective: We investigated the accuracy of host markers detected in Mtb antigen-stimulated whole blood culture supernatant in the diagnosis of TB., Methods: Prospectively, blood from 322 individuals with presumed TB disease from six African sites was stimulated with four different Mtb antigens (Rv0081, Rv1284, ESAT-6/CFP-10, and Rv2034) in a 24 h whole blood stimulation assay (WBA). The concentrations of 42 host markers in the supernatants were measured using the Luminex multiplex platform. Diagnostic biosignatures were investigated through the use of multivariate analysis techniques., Results: 17% of the participants were HIV infected, 106 had active TB disease and in 216 TB was excluded. Unstimulated concentrations of CRP, SAA, ferritin and IP-10 had better discriminating ability than markers from stimulated samples. Accuracy of marker combinations by general discriminant analysis (GDA) identified a six analyte model with 77% accuracy for TB cases and 84% for non TB cases, with a better performance in HIV uninfected patients., Conclusions: A biosignature of 6 cytokines obtained after stimulation with four Mtb antigens has moderate potential as a diagnostic tool for pulmonary TB disease individuals and stimulated marker expression had no added value to unstimulated marker performance., (Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Background: User-friendly, rapid, inexpensive yet accurate TB diagnostic tools are urgently needed at points of care in resource-limited settings. We investigated host biomarkers detected in serum samples obtained from adults with signs and symptoms suggestive of TB at primary healthcare clinics in five African countries (Malawi, Namibia, South Africa, The Gambia and Uganda), for the diagnosis of TB disease., Methods: We prospectively enrolled individuals presenting with symptoms warranting investigation for pulmonary TB, prior to assessment for TB disease. We evaluated 22 host protein biomarkers in stored serum samples using a multiplex cytokine platform. Using a pre-established diagnostic algorithm comprising of laboratory, clinical and radiological findings, participants were classified as either definite TB, probable TB, questionable TB status or non-pulmonary TB., Results: Of the 716 participants enrolled, 185 were definite and 29 were probable TB cases, 6 had questionable TB disease status, whereas 487 had no evidence of TB. A seven-marker biosignature of C reactive protein, transthyretin, IFN-γ, complement factor H, apolipoprotein-A1, inducible protein 10 and serum amyloid A identified on a training sample set (n=491), diagnosed TB disease in the test set (n=210) with sensitivity of 93.8% (95% CI 84.0% to 98.0%), specificity of 73.3% (95% CI 65.2% to 80.1%), and positive and negative predictive values of 60.6% (95% CI 50.3% to 70.1%) and 96.4% (95% CI 90.5% to 98.8%), respectively, regardless of HIV infection status or study site., Conclusions: We have identified a seven-marker host serum protein biosignature for the diagnosis of TB disease irrespective of HIV infection status or ethnicity in Africa. These results hold promise for the development of a field-friendly point-of-care screening test for pulmonary TB., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Epidemiological differences exist between Mycobacterium africanum (Maf)- and Mycobacterium tuberculosis (Mtb)-infected patients, but to date, contributing host factors have not been characterised. We analysed clinical outcomes, as well as soluble markers and gene expression profiles in unstimulated, and ESAT6/CFP-10-, whole-Maf- and Mtb-stimulated blood samples of 26 Maf- and 49 Mtb-HIV-negative tuberculosis patients before, and after 2 and 6 months of anti-tuberculosis therapy. Before treatment, both groups had similar clinical parameters, but differed in few cytokines concentration and gene expression profiles. Following treatment the body mass index, skinfold thickness and chest X-ray scores showed greater improvement in the Mtb- compared to Maf-infected patients, after adjusting for age, sex and ethnicity (p = 0.02; 0.04 and 0.007, respectively). In addition, in unstimulated blood, IL-12p70, IL12A and TLR9 were significantly higher in Maf-infected patients, while IL-15, IL-8 and MIP-1α were higher in Mtb-infected patients. Overnight stimulation with ESAT-6/CFP-10 induced significantly higher levels of IFN-γ and TNF-α production, as well as gene expression of CCL4, IL1B and TLR4 in Mtb- compared to Maf-infected patients. Our study confirms differences in clinical features and immune genes expression and concentration of proteins associated with inflammatory processes between Mtb- and Maf-infected patients following anti-tuberculosis treatment These findings have public health implications for treatment regimens, and biomarkers for tuberculosis diagnosis and susceptibility.

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., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Tuberculosis is one of the leading causes of morbidity and mortality in developing countries. Analysis of the host immune response may help with generating point-of-care tests for personalised monitoring. Thus, the aim of this study was to assess the relationship between immune activation markers: C-reactive protein (CRP), Beta2 microglobulin (B2M) and Neopterin, disease severity prior to treatment and response to therapy in adult pulmonary TB patients., Methods: HIV negative adult pulmonary TB index cases (n = 91) were recruited from the TB clinic at MRC, The Gambia. Plasma samples were collected at enrolment and at 2 and 6 months following TB treatment initiation. An enzyme linked immunosorbent assay (ELISA) was performed for evaluation of CRP, B2M and Neopterin levels and correlated with clinical and microbiological parameters including strain of infection. Disease severity was determined using Chest X-ray (CXR), Body Mass Index (BMI) and sputum smear grade., Results: Plasma levels of all three markers were highly elevated in patients at recruitment and declined significantly during TB therapy. No correlation with disease severity was seen at recruitment. CRP showed the most significant decrease by 2 months of treatment (p < 0.0001) whereas levels of B2M and Neopterin showed little change by 2 months but a significant decrease by 6 months of treatment (p = 0.0002 and p < 0.0001 respectively). At recruitment, B2M levels were significantly higher in subjects infected with Mycobacterium africanum (Maf) compared with those infected with Mycobacterium tuberculosis sensu stricto (Mtb) (p = 0.0075). In addition, while CRP and Neopterin showed a highly significant decline post-treatment regardless of strain (p < 0.0001 for all), B2M showed differential decline depending on strain (p = 0.0153 for Mtb and p = 0.0048 for Maf) and levels were still significantly higher at 6 months in Maf compared to Mtb infected subjects (p = 0.0051)., Conclusion: Our findings suggest that activation markers, particularly CRP, may have a role in identifying good response to TB therapy regardless of the strain of infection and could be further developed as point-of-care tests. In addition, B2M levels may allow differentiation between Mtb and Maf-infected subjects.

Background: Diagnosis of tuberculosis (TB) remains difficult, particularly in resource-limited settings. The development of nucleic acid-based tests for detection of Mycobacterium tuberculosis complex (MTBC) has significantly increased sensitivity compared to conventional smear microscopy and provides results within a matter of hours compared to weeks for the current gold-standard, liquid culture., Methods: In this study we performed side-by-side comparison of mycobacterial detection assays on sputum samples from 285 subjects presenting with symptoms suggestive of TB in The Gambia and a cross-sectional cohort of 156 confirmed TB patients with a median of 2 months of treatment. A novel assay, Loop-Mediated Amplification test for TB (TB-LAMP), was compared to smear microscopy, MGIT culture and GeneXpert MTB/RIF for all samples., Results: When culture was used as the reference standard, we found an overall sensitivity for TB-LAMP of 99% (95% CI: 94.5-99.8) and specificity of 94% (95% CI: 89.3-96.7). When latent class analysis was performed, TB-LAMP had 98.6% (95% CI: 95.9-100) sensitivity and 99% (95% CI: 98.2-100) specificity compared to 91.1% (95% CI: 86.1-96) sensitivity and 100% (95% CI: 98.2-100) specificity for MGIT culture. GeneXpert had the highest sensitivity 99.1% (95% CI: 97.1-100) but the lowest specificity 96% (95% CI: 92.6-98.3). Both TB-LAMP and GeneXpert showed high sensitivity and specificity regardless of age or strain of infection., Conclusion: Our findings show the diagnostic utility of both GeneXpert and TB-LAMP in The Gambia. Whilst TB-LAMP requires less infrastructure, it is unable to detect drug-resistant patterns and therefore would be most suitable as a screening test for new TB cases in peripheral health clinics., (Copyright © 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)