SUNScholar - Browsing by Author "Walzl, Gerhard"

Browsing by Author "Walzl, Gerhard"

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Africa-wide evaluation of host biomarkers in QuantiFERON supernatants for the diagnosis of pulmonary tuberculosis
(Nature Research, 2018-02-08) Chegou, Novel N.; Sutherland, Jayne S.; Namuganga, Anna-Ritah; Corstjens, Paul L. A. M.; Geluk, Annemieke; Gebremichael, Gebremedhin; Mendy, Joseph; Malherbe, Stephanus; Stanley, Kim; Van Der Spuy, Gian D.; Kriel, Magdalena; Loxton, Andre G.; Kriel, Belinda; Simukonda, Felanji; Bekele, Yonas; Sheehama, Jacob A.; Nelongo, Josefina; Van Der Vyver, Marieta; Gebrexabher, Atsbeha; Hailu, Habteyes; Esterhuyse, Maria M.; Rosenkrands, Ida; Aagard, Claus; Kidd, Martin; Kassa, Desta; Mihret, Adane; Howe, Rawleigh; Cliff, Jacqueline M.; Crampin, Amelia C.; Mayanja-Kizza, Harriet; Kaufmann, Stefan H. E.; Dockrell, Hazel M.; Ottenhoff, Tom H. M.; Walzl, Gerhard; AE-TBC consortium
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.
Analysis of host responses to secreted, latent and reactivation Mycobacterium tuberculosis antigens in a multi-site study of subjects with different TB and HIV infection states in sub-Saharan Africa
(Public Library of Science, 2013-09-10) Sutherland, Jayne S.; Lalor, Maeve K.; Black, Gillian F.; Ambrose, Lyn R.; Loxton, Andre G.; Chegou, Novel N.; Kassa, Desta; Mihret, Adane; Howe, Rawleigh; Mayanja-Kizza, Harriet; Gomez, Marie P.; Donkor, Simon; Franken, Kees; Boom, W. Henry; Thiel, Bonnie A.; Crampin, Amelia C.; Hanekom, Willem; Klein, Michel R.; Parida, Shreemanta K.; Ota, Martin; Walzl, Gerhard; Ottenhoff, Tom H. M.; Dockrell, Hazel M.; Kaufmann, Stefan H. E.
Background: Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas. Methods: We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens. Results: There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST- and TST+ contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST+ contacts (LTBI) compared to TB and TST- contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen. Conclusions: Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials.
Application of cerebrospinal fluid host protein biosignatures in the diagnosis of tuberculous meningitis in children from a high burden setting
(Hindawi, 2019-04) Manyelo, Charles M.; Solomons, Regan; Snyders, Candice I.; Manngo, Portia M.; Mutavhatsindi, Hygon; Kriel, Belinda; Stanley, Kim; Walzl, Gerhard; Chegou, Novel N.
Background. The diagnosis of tuberculous meningitis (TBM) especially in children is challenging. New tests are urgently needed for the diagnosis of the disease, especially in resource-limited settings. Methods. We collected cerebrospinal fluid (CSF) samples from children presenting with symptoms requiring investigation for meningitis at a tertiary hospital in Cape Town, South Africa. Children were later classified as TBM or no TBM using published case definitions. Using a multiplex platform, we investigated the concentrations of biomarkers comprising a previously established 3-marker biosignature (VEGF, IL-13, and LL-37) and other potentially useful host biomarkers as diagnostic candidates for TBM. Findings. Out of 47 children, age, 3 months to 13 years, 23 were diagnosed with TBM and six (16%) were HIV-infected. We validated the previously identified CSF biosignature (sensitivity of 95.7% (95% CI, 79.0-99.2%) and specificity of 37.5% (95% CI, 21.2-57.3%)). However, substitution of IL-13 and LL-37 with IFN-γ and MPO, respectively, resulted in improved accuracy (area under the ROC curve (AUC) = 0 97, 95% CI, 0.92-1.00, up to 91.3% (21/23) sensitivity and up to 100% (24/24) specificity). An alternative four-marker biosignature (sICAM-1, MPO, CXCL8, and IFN-γ) also showed potential, with an AUC of 0.97. Conclusion. We validated a previously identified CSF biosignature and showed that refinement of this biosignature by incorporation of other biomarkers diagnosed TBM with high accuracy. Incorporation of these biomarkers into a point-of-care or bedside diagnostic test platform may result in the improved management of TBM in children.
Bacterial loads measured by the Xpert MTB/RIF assay as markers of culture conversion and bacteriological cure in pulmonary TB
(Public Library of Science, 2016) Shenai, Shubhada; Ronache, Katharina; Malherbe, Stefanus; Stanley, Kim; Kriel, Magdalena; Winter, Jill; Peppard, Thomas; Barry, Charles E.; Wang, Jing; Dodd, Lori E.; Via, Laura E.; Barry, Clifton E. 3rd; Walzl, Gerhard; Alland, David
Introduction: Biomarkers are needed to monitor tuberculosis (TB) treatment and predict treatment outcomes. We evaluated the Xpert MTB/RIF (Xpert) assay as a biomarker for TB treatment during and at the end of the 24 weeks therapy. Methods: Sputum from 108 HIV-negative, culture-positive pulmonary TB patients was analyzed using Xpert at time points before and during anti-TB therapy. Results were compared against culture. Direct Xpert cycle-threshold (Ct), a change in the Ct (delta Ct), or a novel “percent closing of baseline Ct deficit” (percent closing) were evaluated as classifiers of same-day and end-of-treatment culture and therapeutic outcomes. Results: Xpert was positive in 29/95 (30.5%) of subjects at week 24; and positive one year after treatment in 8/64 (12.5%) successfully-treated patients who remained free of tuberculosis. We identified a relationship between initial bacterial load measured by baseline Xpert Ct and time to culture conversion (hazard ratio 1.06, p = 0.0023), and to the likelihood of being among the 8 treatment failures at week 24 (AUC = 72.8%). Xpert Ct was even more strongly associated with culture conversion on the day the test was performed with AUCs 96.7%, 99.2%, 86.0% and 90.2%, at Day 7, Week 4, 8 and 24, respectively. Compared to baseline Ct measures alone, a combined measure of baseline Ct plus either Delta Ct or percent closing improved the classification of treatment failure status to a 75% sensitivity and 88.9% specificity. Conclusions: Genome loads measured by Xpert provide a potentially-useful biomarker for classifying same day culture status and predicting response to therapy.
Baseline predictors of sputum culture conversion in pulmonary tuberculosis : importance of cavities, smoking, time to detection and W-Beijing genotype
(PLOS, 2012-01-04) Visser, Marianne E.; Stead, Michael C.; Walzl, Gerhard; Warren, Rob; Schomaker, Michael; Grewal, Harleen M. S.; Swart, Elizabeth C.; Maartens, Gary
Background: Time to detection (TTD) on automated liquid mycobacterial cultures is an emerging biomarker of tuberculosis outcomes. The M. tuberculosis W-Beijing genotype is spreading globally, indicating a selective advantage. There is a paucity of data on the association between baseline TTD and W-Beijing genotype and tuberculosis outcomes. Aim: To assess baseline predictors of failure of sputum culture conversion, within the first 2 months of antitubercular therapy, in participants with pulmonary tuberculosis. Design: Between May 2005 and August 2008 we conducted a prospective cohort study of time to sputum culture conversion in ambulatory participants with first episodes of smear and culture positive pulmonary tuberculosis attending two primary care clinics in Cape Town, South Africa. Rifampicin resistance (diagnosed on phenotypic susceptibility testing) was an exclusion criterion. Sputum was collected weekly for 8 weeks for mycobacterial culture on liquid media (BACTEC MGIT 960). Due to missing data, multiple imputation was performed. Time to sputum culture conversion was analysed using a Cox-proportional hazards model. Bayesian model averaging determined the posterior effect probability for each variable. Results: 113 participants were enrolled (30.1% female, 10.5% HIV-infected, 44.2% W-Beijing genotype, and 89% cavities). On Kaplan Meier analysis 50.4% of participants underwent sputum culture conversion by 8 weeks. The following baseline factors were associated with slower sputum culture conversion: TTD (adjusted hazard ratio (aHR) = 1.11, 95% CI 1.02; 1.2), lung cavities (aHR = 0.13, 95% CI 0.02; 0.95), ever smoking (aHR = 0.32, 95% CI 0.1; 1.02) and the W-Beijing genotype (aHR = 0.51, 95% CI 0.25; 1.07). On Bayesian model averaging, posterior probability effects were strong for TTD, lung cavitation and smoking and moderate for W-Beijing genotype. Conclusion: We found that baseline TTD, smoking, cavities and W-Beijing genotype were associated with delayed 2 month sputum culture. Larger studies are needed to confirm the relationship between the W-Beijing genotype and sputum culture conversion.
Biomarkers of inflammation, immunosuppression and stress with active disease are revealed by metabolomic profiling of tuberculosis patients
(Public Library of Science, 2012-07-23) Weiner, January; Parida, Shreemanta K.; Maertzdorf, Jeroen; Black, Gillian F.; Repsilber, Dirk; Telaar, Anna; Mohney, Robert P.; Arndt-Sullivan, Cordelia; Ganoza, Christian A.; Fae, Kellen C.; Walzl, Gerhard; Kaufmann, Stefan H. E.
Although tuberculosis (TB) causes more deaths than any other pathogen, most infected individuals harbor the pathogen without signs of disease. We explored the metabolome of >400 small molecules in serum of uninfected individuals, latently infected healthy individuals and patients with active TB. We identified changes in amino acid, lipid and nucleotide metabolism pathways, providing evidence for anti-inflammatory metabolomic changes in TB. Metabolic profiles indicate increased activity of indoleamine 2,3 dioxygenase 1 (IDO1), decreased phospholipase activity, increased abundance of adenosine metabolism products, as well as indicators of fibrotic lesions in active disease as compared to latent infection. Consistent with our predictions, we experimentally demonstrate TB-induced IDO1 activity. Furthermore, we demonstrate a link between metabolic profiles and cytokine signaling. Finally, we show that 20 metabolites are sufficient for robust discrimination of TB patients from healthy individuals. Our results provide specific insights into the biology of TB and pave the way for the rational development of metabolic biomarkers for TB. © 2012 Weiner et al.
A broad profile of co-dominant epitopes shapes the peripheral Mycobacterium tuberculosis specific CD8+ T-Cell immune response in South African patients with active tuberculosis
(Public Library of Science, 2013-03-26) Axelsson-Robertson, Rebecca; Loxton, Andre G.; Walzl, Gerhard; Ehlers, Marthie M.; Kock, Marleen M.; Zumla, Alimuddin; Maeurer, Markus
We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition.
Cardiovascular risk and endothelial function in people living with HIV/AIDS: design of the multi-site, longitudinal EndoAfrica study in the Western Cape Province of South Africa
(BioMed Central, 2017-01-07) Strijdom, Hans; De Boever, Patrick; Walzl, Gerhard; Essop, M. Faadiel; Nawrot, Tim S.; Webster, Ingrid; Westcott, Corli; Mashele, Nyiko; Everson, Frans; Malherbe, Stephanus T.; Stanley, Kim; Kessler, Harald H.; Stelzl, Evelyn; Goswami, Nandu
Background: There is growing evidence of an interaction between HIV-infection, anti-retroviral therapy (ART) and cardiovascular diseases (CVD). Epidemiological studies in Europe and North America have been observing a shift towards an increased incidence of coronary heart disease and acute myocardial infarctions in HIV-infected populations compared to the general population even after adjusting for traditional cardiovascular risk factors. Despite South Africa (and sub-Saharan Africa, SSA) being regarded as the epicentre of the global HIV epidemic, very little is known about the prevalence of cardiovascular risk factors and precursors of vascular disease in HIV-infected populations in this region. The knowledge gap is further widened by the paucity of data from prospective studies. We present the rationale, objectives and key methodological features of the EndoAfrica study, which aims to determine whether HIVinfection and ART are associated with altered cardiovascular risk and changes in vascular endothelial structure and function in adults living in the Western Cape Province of South Africa. Methods: In this longitudinal study, comprehensive cardiovascular assessments of HIV-negative and HIV-positive (with and without ART) study participants are performed by clinical and biochemical screening for traditional cardiovascular risk factors and biomarkers of CVD. Vascular and endothelial function is determined by brachial artery flow-mediated dilatation (FMD), carotid-intima-thickness (IMT) measurements and quantitative retinal blood vessel analyses, complemented by vascular endothelial biomarker assays. Finally, we aim to statistically determine whether HIVinfection and/or ART are associated with increased cardiovascular risk and vascular endothelial dysfunction, and determine whether there is progression/regression in these endpoints 18 months after the baseline assessments. Discussion: The EndoAfrica study provides a unique opportunity to recruit a cohort of HIV-infected patients and HIVnegative controls who will be comprehensively and longitudinally assessed for cardiovascular risk and disease profile with vascular endothelial function as a potentially important intermediate cardiovascular phenotype. To our knowledge, it is the first time that such a systematic study has been established in the context of SSA and South Africa.
Caveolin-1 controls vesicular TLR2 expression, p38 signaling and T cell suppression in BCG infected Murine Monocytic myeloid-derived suppressor cells
(Frontiers Media, 2019) John, Vini; Kotze, Leigh A.; Ribechini, Eliana; Walzl, Gerhard; Du Plessis, Nelita; Lutz, Manfred B.
Monocytic myeloid-derived suppressor cells (M-MDSCs) and granulocytic MDSCs (G-MDSCs) have been found to be massively induced in TB patients as well in murine Mtb infection models. However, the interaction of mycobacteria with MDSCs and its role in TB infection is not well studied. Here, we investigated the role of Cav-1 for MDSCs infected with Mycobacterium bovis Bacille-Calmette-Guerín (BCG). MDSCs that were generated from murine bone marrow (MDSCs) of wild-type (WT) or Cav1−/− mice upregulated Cav-1, TLR4 and TLR2 expression after BCG infection on the cell surface. However, Cav-1 deficiency resulted in a selective defect of intracellular TLR2 levels predominantly in the M-MDSC subset. Further analysis indicated no difference in the phagocytosis of BCG by M-MDSCs from WT and Cav1−/− mice or caveosome formation, but a reduced capacity to up-regulate surface markers, to secrete various cytokines, to induce iNOS and NO production required for suppression of T cell proliferation, whereas Arg-1 was not affected. Among the signaling pathways affected by Cav-1 deficiency, we found lower phosphorylation of the p38 mitogen-activated protein kinase (MAPK). Together, our findings implicate that (i) Cav-1 is dispensable for the internalization of BCG, (ii) vesicular TLR2 signaling in M-MDSCs is a major signaling pathway induced by BCG, (iii) vesicular TLR2 signals are controlled by Cav-1, (iv) vesicular TLR2/Cav-1 signaling is required for T cell suppressor functions.
CCL1 and IL-2Ra differentiate Tuberculosis disease from latent infection Irrespective of HIV infection in low TB burden countries
(ElsevierLtd, 2021-10) Chendi, Bih H.; Tveiten, Hallgeir; Snyders, Candice I.; Tonby, Kristian; Jenum, Synne; Nielsen, Susanne Dam; Hove-Skovsgaard, Malene; Walzl, Gerhard; Chegou, Novel N.; Dyrhol-Riise, Anne M
Objectives: To evaluate the performance of selected host immunological biomarkers in differentiating tu- berculosis (TB) disease from latent TB infection (LTBI) in HIV uninfected and infected individuals enrolled in TB low-burden countries. Design: Participants with TB disease ( N = 85) and LTBI ( N = 150) were recruited from prospective co- horts at hospitals in Norway and Denmark. Plasma concentrations of 54 host markers were assessed by Luminex multiplex immunoassays. Using receiver operator characteristic curves and general discriminant analysis, we determined the abilities of individual and combined biomarkers to discriminate between TB disease and LTBI including when patients were stratified according to HIV infection status. Results: Regardless of the groups compared, CCL1 and IL-2Ra were the most accurate single biomarkers in differentiating TB disease from LTBI. Regardless of HIV status, a 4-marker signature (CCL1 + RANTES + CRP + MIP-1 α) derived from a training set ( n = 155) differentiated TB disease from LTBI in the test set ( n = 67) with a sensitivity of 56.0% (95% CI, 34.9–75.6) and a specificity of 85.7% (95% CI, 71.5–94.6). A 5-marker signature derived from the HIV uninfected group (CCL1 + RANTES + MIP- 1 α+ procalcitonin + IP-10) performed in HIV-infected individuals with a sensitivity of 75.0% and a speci- ficity of 96.7% after leave-one-out cross validation. A 2-marker signature (CCL1 + TNF- α) identified in HIV- infected persons performed in HIV-uninfected with a sensitivity and specificity of 66.7% and 100% respec- tively in the test set. Conclusions: Plasma CCL1 and IL-2Ra have potential as biomarkers for differentiating TB disease from LTBI in low TB burden settings unaffected by HIV infection. Combinations between these and other biomarkers in bio-signatures for global use warrant further exploration.
Changes in host immune–endocrine relationships during tuberculosis treatment in patients with cured and failed treatment outcomes
(Frontiers Media, 2017) Kleynhans, Leanie; Ruzive, Sheena; Ehlers, Lizaan; Thiart, Lani; Chegou, Novel N.; Conradie, Magda; Kriel, Magdalena; Kim Stanley; Van Der Spuy, Gian D.; Kidd, Martin; Van Helden; Walzl, Gerhard; Ronacher, Katharina
A bidirectional communication between the immune and endocrine systems exists and facilitates optimum responses in the host during infections. This is in part achieved through changes in secretion patterns of hypothalamic hormones induced by inflammatory cytokines. The aim of this study was to elucidate the immune–endocrine alterations during tuberculosis (TB) treatment in patients with cured and failed TB treatment outcomes. Blood samples were collected from 27 cured and 10 failed patients and hormone as well as cytokine concentrations quantified at baseline, week 4, and month 6 of TB treatment. Hormone profiles of the two treatment outcome groups were different from each other prior to as well as during TB treatment. Treatment response effects were observed for cortisol, estradiol, T3, T4 ghrelin, leptin, amylin, adiponectin, and dehydroepiandrosterone (DHEA). Trends suggest that T4, amylin, and DHEA concentrations were different between treatment outcomes, although these did not reach statistical significance. Relationships between endocrine and inflammatory markers and the biological pathways involved differed between cured and failed treatment patients. These results highlight the complex interaction between the endocrine and immune system during active TB disease and throughout treatment and suggest that endocrine markers in conjunction with inflammatory markers may be useful in predicting unfavorable treatment outcomes.
Combination of gene expression patterns in whole blood discriminate between tuberculosis infection states
(BioMed Central, 2014-05) Mihret, Adane; Loxton, Andre G.; Bekele, Yonas; Kaufmann, Stefan H. E.; Kidd, Martin; Haks, Marielle C.; Ottenhoff, Tom H. M.; Aseffa, Abraham; Howe, Rawleigh; Walzl, Gerhard
Background Genetic factors are involved in susceptibility or protection to tuberculosis (TB). Apart from gene polymorphisms and mutations, changes in levels of gene expression, induced by non-genetic factors, may also determine whether individuals progress to active TB. Methods We analysed the expression level of 45 genes in a total of 47 individuals (23 healthy household contacts and 24 new smear-positive pulmonary TB patients) in Addis Ababa using a dual colour multiplex ligation-dependent probe amplification (dcRT-MLPA) technique to assess gene expression profiles that may be used to distinguish TB cases and their contacts and also latently infected (LTBI) and uninfected household contacts. Results The gene expression level of BLR1, Bcl2, IL4d2, IL7R, FCGR1A, MARCO, MMP9, CCL19, and LTF had significant discriminatory power between sputum smear-positive TB cases and household contacts, with AUCs of 0.84, 0.81, 0.79, 0.79, 0.78, 0.76, 0.75, 0.75 and 0.68 respectively. The combination of Bcl2, BLR1, FCGR1A, IL4d2 and MARCO identified 91.66% of active TB cases and 95.65% of household contacts without active TB. The expression of CCL19, TGFB1, and Foxp3 showed significant difference between LTBI and uninfected contacts, with AUCs of 0.85, 0.82, and 0.75, respectively, whereas the combination of BPI, CCL19, FoxP3, FPR1 and TGFB1 identified 90.9% of QFT- and 91.6% of QFT+ household contacts. Conclusions Expression of single and especially combinations of host genes can accurately differentiate between active TB cases and healthy individuals as well as between LTBI and uninfected contacts.
Complement component C1q as serum biomarker to detect active tuberculosis
(Frontiers Media, 2018) Lubbers, Rosalie; Sutherland, Jayne S.; Goletti, Delia; De Paus, Roelof A.; Van Moorsel, Coline H. M.; Veltkamp, Marcel; Vestjens, Stefan M. T.; Bos, Willem J. W.; Petrone, Linda; Del Nonno, Franca; Bajema, Ingeborg M.; Dijkman, Karin; Verreck, Frank A. W.; Walzl, Gerhard; Gelderman, Kyra A.; Groeneveld, Geert H.; Geluk, Annemieke; Ottenhoff, Tom H. M.; Joosten, Simone A.; Trouw, Leendert A.
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.
Decreased expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4+ T cells and peripheral blood from tuberculosis patients
(Public Library of Science, 2013-04-16) Kleinsteuber, Katja; Heesch, Kerrin; Schattling, Stefanie; Kohns, Malte; Sander-Julch, Claudia; Walzl, Gerhard; Hesseling, Anneke; Mayatepek, Ertan; Fleischer, Bernhard; Marx, Florian M.; Jacobsen, Marc
The vast majority of Mycobacterium tuberculosis (M. tuberculosis) infected individuals are protected from developing tuberculosis and T cells are centrally involved in this process. MicroRNAs (miRNA) regulate T-cell functions and are biomarker candidates of disease susceptibility and treatment efficacy in M. tuberculosis infection. We determined the expression profile of 29 selected miRNAs in CD4+ T cells from tuberculosis patients and contacts with latent M. tuberculosis infection (LTBI). These analyses showed lower expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4+ T cells from tuberculosis patients. Whole blood miRNA candidate analyses verified decreased expression of miR-26a, miR-29a, and miR-142-3p in children with tuberculosis as compared to healthy children with LTBI. Despite marked variances between individual donor samples, trends of increased miRNA candidate expression during treatment and recovery were observed. Functional in vitro analysis identified increased miR-21 and decreased miR-26a expression after re-stimulation of T cells. In vitro polarized Interleukin-17 positive T-cell clones showed activation-dependent miR-29a up-regulation. In order to characterize the role of miR-29a (a described suppressor of Interferon-γ in tuberculosis), we analyzed M. tuberculosis specific Interferon-γ expressing T cells in children with tuberculosis and healthy contacts but detected no correlation between miR-29a and Interferon-γ expression. Suppression of miR-29a in primary human T cells by antagomirs indicated no effect on Interferon-γ expression after in vitro activation. Finally, classification of miRNA targets revealed only a moderate overlap between the candidates. This may reflect differential roles of miR-21, miR-26a, miR-29a, and miR-142-3p in T-cell immunity against M. tuberculosis infection and disease.
Detection of a combination of serum IgG and IgA antibodies against selected mycobacterial targets provides promising diagnostic signatures for active TB
(Impact Journals, 2017-03-24) Awoniyi, Dolapo O.; Baumann, Ralf; Chegou, Novel N.; Kriel, Belinda; Jacobs, Ruschca; Kidd, Martin; Loxton, Andre G.; Kaempfer, Susanne; Singh, Mahavir; Walzl, Gerhard
ENGLISH ABSTRACT: Immunoglobulin G (IgG) based tests for the diagnosis of active tuberculosis (TB) disease often show a lack of specificity in TB endemic regions, which is mainly due to a high background prevalence of LTBI. Here, we investigated the combined performance of the responses of different Ig classes to selected mycobacterial antigens in primary healthcare clinic attendees with signs and symptoms suggestive of TB. The sensitivity and specificity of IgA, IgG and/or IgM to LAM and 7 mycobacterial protein antigens (ESAT-6, Tpx, PstS1, AlaDH, MPT64, 16kDa and 19kDa) and 2 antigen combinations (TUB, TB-LTBI) in the plasma of 63 individuals who underwent diagnostic work-up for TB after presenting with symptoms and signs compatible with possible active TB were evaluated. Active TB was excluded in 42 individuals of whom 21 has LTBI whereas active TB was confirmed in 21 patients of whom 19 had a follow-up blood draw at the end of 6-month anti-TB treatment. The leading single serodiagnostic markers to differentiate between the presence or absence of active TB were anti-16 kDa IgA, anti-MPT64 IgA with sensitivity and specificity of 90%/90% and 95%/90%, respectively. The combined use of 3 or 4 antibodies further improved this performance to accuracies above 95%. After successful completion of anti-TB treatment at month 6, the levels of 16 kDa IgA and 16 kDa IgM dropped significantly whereas LAM IgG and TB-LTBI IgG increased. These results show the potential of extending investigation of anti-tuberculous IgG responses to include IgM and IgA responses against selected protein and non-protein antigens in differentiating active TB from other respiratory diseases in TB endemic settings.
Detection of tuberculosis recurrence, diagnosis and treatment response by a blood transcriptomic risk signature in HIV-infected persons on antiretroviral therapy
(Frontiers Media, 2019) Darboe, Fatoumatta; Mbandi, Stanley Kimbung; Naidoo, Kogieleum; Yende-Zuma, Nonhlanhla; Lewis, Lara; Thompson, Ethan G.; Duffy, Fergal J.; Fishe, Michelle; Filander, Elizabeth; Van Rooyen, Michele; Bilek, Nicole; Mabwe, Simbarashe; McKinnon, Lyle R.; Chegou, Novel; Loxton, Andre; Walzl, Gerhard; Tromp, Gerard; Padayatchi, Nesri; Govender, Dhineshree; Hatherill, Mark; Karim, Salim Abdool; Zak, Daniel E.; Penn-Nicholson, Adam; Scriba, Thomas J.; SATVI Clinical Immunology Team
ENGLISH ABSTRACT: HIV-infected individuals are at high risk of tuberculosis disease and those with prior tuberculosis episodes are at even higher risk of disease recurrence. A non-sputum biomarker that identifies individuals at highest tuberculosis risk would allow targeted microbiological testing and appropriate treatment and also guide need for prolonged therapy. We determined the utility of a previously developed whole blood transcriptomic correlate of risk (COR) signature for (1) predicting incident recurrent tuberculosis, (2) tuberculosis diagnosis and (3) its potential utility for tuberculosis treatment monitoring in HIV-infected individuals. We retrieved cryopreserved blood specimens from three previously completed clinical studies and measured the COR signature by quantitative microfluidic real-time-PCR. The signature differentiated recurrent tuberculosis progressors from non-progressors within 3 months of diagnosis with an area under the Receiver-operating characteristic (ROC) curve (AUC) of 0.72 (95% confidence interval (CI), 0.58–0.85) amongst HIV-infected individuals on antiretroviral therapy (ART). Twenty-five of 43 progressors (58%) were asymptomatic at microbiological diagnosis and thus had subclinical disease. The signature showed excellent diagnostic discrimination between HIV-uninfected tuberculosis cases and controls (AUC 0.97; 95%CI 0.94–1). Performance was lower in HIV-infected individuals (AUC 0.83; 95%CI 0.81–0.96) and signature scores were directly associated with HIV viral loads. Tuberculosis treatment response in HIV-infected individuals on ART with a new recurrent tuberculosis diagnosis was also assessed. Signature scores decreased significantly during treatment. However, pre-treatment scores could not differentiate between those who became sputum negative before and after 2 months. Direct application of the unmodified blood transcriptomic COR signature detected subclinical and active tuberculosis by blind validation in HIV-infected individuals. However, prognostic performance for recurrent tuberculosis, and performance as diagnostic and as treatment monitoring tool in HIV-infected persons was inferior to published results from HIV-negative cohorts. Our results suggest that performance of transcriptomic signatures comprising interferon stimulated genes are negatively affected in HIV-infected individuals, especially in those with incompletely suppressed viral loads.
Diabetes mellitus among pulmonary tuberculosis patients from 4 tuberculosis-endemic countries : the TANDEM study
(Oxford University Press, 2019-04) Ugarte-Gil, Cesar; Alisjahbana, Bachti; Ronacher, Katharina; Lelia Riza, Anca; Koesoemadinata, Raspati C.; Malherbe, Stephanus T.; Cioboata, Ramona; Llontop, Juan Carlos; Kleynhans, Leanie; Lopez, Sonia; Santoso, Prayudi; Marius, Ciontea; Villaizan, Katerine; Ruslami, Rovina; Walzl, Gerhard; Panduru, Nicolae Mircea; Dockrell, Hazel M.; Hill, Philip C.; Allister, Susan Mc; Pearson, Fiona; Moore, David A. J.; Critchley, Julia A.; van Crevel, Reinout
Background Diabetes mellitus (DM) increases active tuberculosis (TB) risk and worsens TB outcomes, jeopardizing TB control especially in TB-endemic countries with rising DM prevalence rates. We assessed DM status and clinical correlates in TB patients across settings in Indonesia, Peru, Romania, and South Africa. Methods Age-adjusted DM prevalence was estimated using laboratory glycated hemoglobin (HbA1c) or fasting plasma glucose in TB patients. Detailed and standardized sociodemographic, anthropometric, and clinical measurements were made. Characteristics of TB patients with or without DM were compared using multilevel mixed-effect regression models with robust standard errors. Results Of 2185 TB patients (median age 36.6 years, 61.2% male, 3.8% human immunodeficiency virus–infected), 12.5% (267/2128) had DM, one third of whom were newly diagnosed. Age-standardized DM prevalence ranged from 10.9% (South Africa) to 19.7% (Indonesia). Median HbA1c in TB–DM patients ranged from 7.4% (Romania) to 11.3% (Indonesia). Compared to those without DM, TB–DM patients were older and had a higher body mass index (BMI) (P value < .05). Compared to those with newly diagnosed DM, TB patients with diagnosed DM had higher BMI and HbA1c, less severe TB, and more frequent comorbidities, DM complications, and hypertension (P value < .05). Conclusions We show that DM prevalence and clinical characteristics of TB–DM vary across settings. Diabetes is primarily known but untreated, hyperglycemia is often severe, and many patients with TB–DM have significant cardiovascular disease risk and severe TB. This underlines the need to improve strategies for better clinical management of combined TB and DM.
Diagnostic potential of novel salivary host biomarkers as candidates for the immunological diagnosis of tuberculosis disease and monitoring of tuberculosis treatment response
(Public Library of Science, 2016-08-03) Jacobs, Ruschca; Maasdorp, Elizna; Malherbe, Stephanus; Loxton, Andre G.; Stanley, Kim; Van Der Spuy, Gian; Walzl, Gerhard; Chegou, Novel N.
Background: There is an urgent need for new tools for the early diagnosis of TB disease and monitoring of the response to treatment, especially in resource-constrained settings. We investigated the usefulness of host markers detected in saliva as candidate biomarkers for the immunological diagnosis of TB disease and monitoring of treatment response. Methods: We prospectively collected saliva samples from 51 individuals that presented with signs and symptoms suggestive of TB disease at a health centre in Cape Town, South Africa, prior to the establishment of a clinical diagnosis. Patients were later classified as having TB disease or other respiratory disease (ORD), using a combination of clinical, radiological and laboratory findings. We evaluated the concentrations of 69 host markers in saliva samples using a multiplex cytokine platform, and assessed the diagnostic potentials of these markers by receiver operator characteristics (ROC) curve analysis, and general discriminant analysis. Results: Out of the 51 study participants, 18 (35.4%) were diagnosed with TB disease and 12 (23.5%) were HIV infected. Only two of the 69 host markers that were evaluated (IL-16 and IL-23) diagnosed TB disease individually with area under the ROC curve ≥0.70. A five-marker biosignature comprising of IL-1β, IL-23, ECM-1, HCC1 and fibrinogen diagnosed TB disease with a sensitivity of 88.9% (95% CI,76.7–99.9%) and specificity of 89.7% (95% CI, 60.4–96.6%) after leave-one-out cross validation, regardless of HIV infection status. Eight-marker biosignatures performed with a sensitivity of 100% (95% CI, 83.2–100%) and specificity of 95% (95% CI, 68.1–99.9%) in the absence of HIV infection. Furthermore, the concentrations of 11 of the markers changed during treatment, indicating that they may be useful in monitoring of TB treatment response. Conclusion: We have identified novel salivary biosignatures which may be useful in the diagnosis of TB disease and monitoring of the response to TB treatment. Our findings require further validation in larger studies before these biosignatures could be considered for point-of-care screening test development.
Differential expression of host biomarkers in saliva and serum samples from individuals with suspected pulmonary tuberculosis
(Hindawi Publishing Corporation, 2013-09) Phalane, Khutso G.; Kriel, Magdalena; Loxton, Andre G.; Menezes, Angela; Stanley, Kim; Van der Spuy, Gian D.; Walzl, Gerhard; Chegou, Novel N.
The diagnosis of tuberculosis remains challenging in individuals with difficulty in providing good quality sputum samples such as children. Host biosignatures of inflammatory markers may be valuable in such cases, especially if they are based on more easily obtainable samples such as saliva. To explore the potential of saliva as an alternative sample in tuberculosis diagnostic/biomarker investigations, we evaluated the levels of 33 hostmarkers in saliva samples fromindividuals presenting with pulmonary tuberculosis symptoms and compared them to those obtained in serum. Of the 38 individuals included in the study, tuberculosis disease was confirmed in 11 (28.9%) by sputum culture. In both the tuberculosis cases and noncases, the levels of most markers were above the minimum detectable limit in both sample types, but there was no consistent pattern regarding the ratio ofmarkers in serum/saliva. Fractalkine, IL-17, IL-6, IL-9, MIP-1𝛽�, CRP, VEGF, and IL-5 levels in saliva and IL-6, IL-2, SAP, and SAA levels in serum were significantly higher in tuberculosis patients (𝑃� < 0.05). These preliminary data indicate that there are significant differences in the levels of host markers expressed in saliva in comparison to those expressed in serumand that inflammatory markers in both sample types are potential diagnostic candidates for tuberculosis disease.
Discovery and validation of a prognostic proteomic signature for tuberculosis progression : a prospective cohort study
(Public Library of Science, 2019) Penn-Nicholson, Adam; Hraha, Thomas; Thompson, Ethan G.; Sterling, David; Mbandi, Stanley Kimbung; Wall, Kirsten M.; Fisher, Michelle; Suliman, Sara; Shankar, Smitha; Hanekom, Willem A.; Janjic, Nebojsa; Hatherill, Mark; Kaufmann, Stefan H. E.; Sutherland, Jayne; Walzl, Gerhard; De Groote, Mary Ann; Ochsne, Urs; Zak, Daniel E.; Scriba, Thomas J.; ACS and GC6–74 cohort study group
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.

Browsing by Author "Walzl, Gerhard"

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