Prospective evaluation of host biomarkers other than interferon gamma in QuantiFERON Plus supernatants as candidates for the diagnosis of tuberculosis in symptomatic individuals

CITATION: Manngo, P. M. et al. 2019. Prospective evaluation of host biomarkers other than interferon gamma in QuantiFERON Plus supernatants as candidates for the diagnosis of tuberculosis in symptomatic individuals. Journal of Infection, 79(3):228-235, doi:10.1016/j.jinf.2019.07.007.The original publication is available at https://www.journalofinfection.comBackground: There is an urgent need for new tools for the diagnosis of TB. We evaluated the usefulness recently described host biomarkers in supernatants from the newest generation of the QuantiFERON test (QuantiFERON Plus) as tools for the diagnosis of active TB. Methods: We recruited individuals presenting at primary health care clinics in Cape Town, South Africa with symptoms requiring investigation for TB disease, prior to the establishment of a clinical diagnosis. Participants were later classified as TB or other respiratory diseases (ORD) based on the results of clinical and laboratory tests. Using a multiplex platform, we evaluated the concentrations of 37 host biomarkers in QuantiFERON Plus supernatants from study participants as tools for the diagnosis of TB. Results: Out of 120 study participants, 35(29.2%) were diagnosed with active TB, 69(57.5%) with ORD whereas 16(13.3%) were excluded. 14(11.6%) of the study participants were HIV infected. Although individ- ual host markers showed potential as diagnostic candidates, the main finding of the study was the identi- fication of a six-marker biosignature in unstimulated supernatants (Apo-ACIII, CXCL1, CXCL9, CCL8, CCL-1, CD56) which diagnosed TB with sensitivity and specificity of 73.9%(95% CI; 51.6–87.8) and 87.6%(95% CI; 77.2–94.5), respectively, after leave-one-out cross validation. Combinations between TB-antigen specific biomarkers also showed potential (sensitivity of 77.3% and specificity of 69.2%, respectively), with multi- ple biomarkers being significantly different between TB patients, Quantiferon Plus Positive and Quantif- eron Plus negative individuals with ORD, regardless of HIV status. Conclusions: Biomarkers detected in QuantiFERON Plus supernatants may contribute to adjunctive diag- nosis of TB.EDCTP , grant no: DRIA2014-311National Research FoundationICIDR (grant no: 5U01IA115619)Publisher's versio

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

Evaluation of host biosignatures for targeted screening for tuberculosis at the point of care, and monitoring of the response to TB treatment

Biomedical Sciences: Molecular Biology and Human Genetic

Characterization of PFF1010c, a type IV Plasmodium fasciparum heat shock protein 40

MSc (Biochemistry)Department of BiochemistryMalaria is caused by protozoa of the genus Plasmodium. Malaria accounts for approximately more than half a million deaths yearly. Of the five species of Plasmodium, P. falciparum accounts for the most deadly form of the disease. P. falciparum survives under various physiological conditions during its life cycle. The parasite employs its molecular chaperones machinery particularly heat shock proteins (Hsps) to protect its protein constituents during physiological stress. Hsps are conserved molecules that constitute a major part of the cell’s molecular chaperone system. P. falciparum Hsps play an important cyto-protective role guaranteeing that the malarial parasite survives under the severe conditions that prevail in the host environment. PFF1010c is a type IV P. falciparum heat shock protein 40. PFF1010c is predicted to be expressed only at the gametocyte stage of the malarial parasite’s life cycle. The aim of the current study was to investigate the expression PFF1010c by parasites and the gametocyte stage as well as characterize the structure-function features of the protein. PFF1010c was successfully expressed in E. coli cells. Despite successful expression of the protein, its purification proved problematic. The attempt to purify PFF1010c was carried out under both native and denaturing conditions. Far Western blot analysis to investigate direct interaction between PFF1010c and PfHsp70-1 was conducted and no interaction was observed. Malarial parasites were harvested at different stages and total protein was isolated. The expression of PFF1010c was confirmed to occur at the gametocyte stage of the parasite’s development using Western blot analysis. This study confirmed that PFF1010c is only expressed at the gametocyte stage of the malarial parasite. Furthermore, PFF1010c was not expressed at the asexual stage. Possible interactors of PFF1010c were predicted by STRING, a bioinformatics based tool. The expression of PfHsp90, PfHop and PfHsp70-1 at the gametocyte stage was investigated and confirmed by Western blot analyses

Potential of host serum protein biomarkers in the diagnosis of tuberculous meningitis in children

CITATION: Manyelo, C. M., et al. 2019. Potential of host serum protein biomarkers in the diagnosis of tuberculous meningitis in children. Frontiers in Pediatrics, 7:376, doi:10.3389/fped.2019.00376.The original publication is available at https://www.frontiersin.orgPublication of this article was funded by the Stellenbosch University Open Access Fund.Background: Tuberculous meningitis (TBM) is the most severe form of tuberculosis and results in high morbidity and mortality in children. Diagnostic delay contributes to the poor outcome. There is an urgent need for new tools for the rapid diagnosis of TBM, especially in children. Methods: We collected serum samples from children in whom TBM was suspected at a tertiary hospital in Cape Town, South Africa. Children were subsequently classified as having TBM or no TBM using a published uniform research case-definition. Using a multiplex cytokine array platform, we investigated the concentrations of serum biomarkers comprising biomarkers that were previously found to be of value in the diagnosis of adult pulmonary TB (CRP, SAA, CFH, IFN-γ, IP-10, Apo-AI, and transthyretin) plus other potentially useful host biomarkers as diagnostic candidates for TBM. Findings: Out of 47 children included in the study, 23 (48.9%) had a final diagnosis of TBM and six were HIV infected. A modified version of the adult 7-marker biosignature in which transthyretin was replaced by NCAM1, diagnosed TBM in children with AUC of 0.80 (95% CI, 0.67–0.92), sensitivity of 73.9% (95% CI, 51.6–89.8%) and specificity of 66.7% (95% CI, 44.7–84.4%), with the other six proteins in the signature (CRP, IFN-γ, IP-10, CFH, Apo-A1, and SAA) only achieving and AUC of 0.75 (95% CI, 0.61–0.90) when used in combination. A new childhood TBM specific 3-marker biosignature (adipsin, Aβ42, and IL-10) showed potential in the diagnosis of TBM, with AUC of 0.84 (95% CI, 0.73–0.96), sensitivity of 82.6% (95 CI, 61.2–95.0%) and specificity of 75.0% (95% CI, 53.3–90.2%) after leave-one-out cross validation. Conclusion: A previously described adult 7-marker serum protein biosignature showed potential in the diagnosis of TBM in children. However, a smaller childhood TBM-specific 3-marker signature demonstrated improved performance characteristics. Our data indicates that blood-based biomarkers may be useful in the diagnosis of childhood TBM and requires further validation in larger cohort studies.https://www.frontiersin.org/articles/10.3389/fped.2019.00376/fullPublisher's versionAuthors retain copyrigh

Application of cerebrospinal fluid host protein biosignatures in the diagnosis of tuberculous meningitis in children from a high burden setting

CITATION: Manyelo, C. M., et al. 2019. Application of cerebrospinal fluid host protein biosignatures in the diagnosis of tuberculous meningitis in children from a high burden setting. Mediators of Inflammation, 2019 (Article ID 7582948), doi:10.1155/2019/7582948.The original publication is available at https://www.hindawi.comPublication of this article was funded by the Stellenbosch University Open Access FundBackground. 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.https://www.hindawi.com/journals/mi/2019/7582948/Publisher's versio