Evaluation of cytokine responses against novel Mtb antigens as diagnostic markers for TB disease.

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

Validation and Optimization of Host Immunological Bio-Signatures for a Point-of-Care Test for TB Disease.

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

Examination of runs of homozygosity in relation to height in an endogamous Namibian population

ObjectivesHeight is a complex, highly heritable polygenic trait subject to both genetic composition and environmental influences. Recent studies suggest that a large proportion of height heritability is determined by the cumulative effect of many low allele frequency variants across the genome. Previous research has also identified an inverse relationship between height and runs of homozygosity (ROH); however, this has yet to be examined within African populations. We aim to identify this association within the Himba, an endogamous Namibian population who are recently bottlenecked, resulting in elevated haplotype sharing and increased homozygosity.Materials and methodsHere, we calculate the fraction of the genome composed of long runs of homozygosity (FROH) in a sample of 245 adults and use mixed effects models to assess its effect on height.ResultsWe find that Himba adults exhibit increased homozygosity. However, in contrast to previous studies in other populations, we do not find a significant effect of FROH on height within the Himba. We further estimated heritability of height, noting both an enrichment of distant relatives and greater developmental homogeneity across households; we find that h g 2 = 0.59 (SE ± 0.146), comparable to estimates reported in Europeans.DiscussionOur results may be due to other environmental variables we were not able to include, measurement error, or low statistical power, but may also imply that phenotypic expression resulting from increased homozygosity may vary from population to population

The ethics and logistics of field-based genetic paternity studies.

The rapidly decreasing costs of generating genetic data sequencing and the ease of new DNA collection technologies have opened up new opportunities for anthropologists to conduct field-based genetic studies. An exciting aspect of this work comes from linking genetic data with the kinds of individual-level traits evolutionary anthropologists often rely on, such as those collected in long-term demographic and ethnographic studies. However, combining these two types of data raises a host of ethical questions related to the collection, analysis and reporting of such data. Here we address this conundrum by examining one particular case, the collection and analysis of paternity data. We are particularly interested in the logistics and ethics involved in genetic paternity testing in the localized settings where anthropologists often work. We discuss the particular issues related to paternity testing in these settings, including consent and disclosure, consideration of local identity and beliefs and developing a process of continued community engagement. We then present a case study of our own research in Namibia, where we developed a multi-tiered strategy for consent and community engagement, built around a double-blind procedure for data collection, analysis and reporting

Multi-center evaluation of a user-friendly lateral flow assay to determine IP-10 and CCL4 levels in blood of TB and non-TB cases in Africa

Objective: Multi-center evaluation of a user-friendly lateral flow test for detection of IP-10 and CCL4 levels in Mycobacterium tuberculosis (Mtb) antigen-stimulated whole blood samples from tuberculosis (TB) suspects. Design and methods: A quantitative lateral flow (LF)-based assay platform was applied to detect chemokines IP-10 and CCL4. Chemokine quantitation was achieved using interference-free, fluorescent up-converting phosphor (UCP) labels. The new assays allowed worldwide shipping and storage without requiring a cold chain and were tested at seven institutes (including Ethiopia, Malawi, The Gambia, South Africa, Uganda and Namibia) employing portable lightweight readers for detection of the UCP label. At each site, clinical samples, confirmed TB and non-TB (i.e. other respiratory diseases (ORD)) cases, were collected and analyzed simultaneously with quality control (QC) human IP-10 or CCL4 standards. Results: Performance of the UCP-LF assay in Africa using QC standards indicated high robustness allowing quantitative detection between 100 and 100,000 pg/mL. The optimized assays allowed successful determination of chemokine levels using 1 μL whole blood sample from the locally recruited subjects with TB or ORD. Conclusion: This African multi-center trial further demonstrated the applicability of the low-tech and robust UCP-LF platform as a convenient quantitative assay for chemokine detection in whole blood. Ambient shipping and storage of all assay reagents and the availability of lightweight standalone readers were acknowledged as essential requirement for test implementation in particular in remote and resource-limited settings

Identifying knowledge needed to improve surgical care in Southern Africa using a theory of change approach

Surgical healthcare has been prioritised in the Southern African Development Community (SADC), a regional intergovernmental entity promoting equitable and sustainable economic growth and socioeconomic development. However, challenges remain in translating political prioritisation into effective and equitable surgical healthcare. The AfroSurg Collaborative (AfroSurg) includes clinicians, public health professionals and social scientists from six SADC countries; it was created to identify context-specific, critical areas where research is needed to inform evidence-grounded policy and implementation. In January 2020, 38 AfroSurg members participated in a theory of change (ToC) workshop to agree on a vision: ‘An African-led, regional network to enable evidence-based, context-specific, safe surgical care, which is accessible, timely, and affordable for all, capturing the spirit of Ubuntu[1]’ and to identify necessary policy and service-delivery knowledge needs to achieve this vision. A unified ToC map was created, and a Delphi survey was conducted to rank the top five priority knowledge needs. In total, 45 knowledge needs were identified; the top five priority areas included (1) mapping of available surgical services, resources and providers; (2) quantifying the burden of surgical disease; (3) identifying the appropriate number of trainees; (4) identifying the type of information that should be collected to inform service planning; and (5) identifying effective strategies that encourage geographical retention of practitioners. Of the top five knowledge needs, four were policy-related, suggesting a dearth of much-needed information to develop regional, evidenced-based surgical policies. The findings from this workshop provide a roadmap to drive locally led research and create a collaborative network for implementing research and interventions. This process could inform discussions in other low-resource settings and enable more evidenced-based surgical policy and service delivery across the SADC countries and beyond

Africa-wide evaluation of host biomarkers in QuantiFERON supernatants for the diagnosis of pulmonary tuberculosis

Abstract 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

Evaluation of cytokine responses against novel Mtb antigens as diagnostic markers for TB disease

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