Browsing by Author "Mutavhatsindi, Hygon"

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    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.
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    Evaluation of host biosignatures for targeted screening for tuberculosis at the point of care, and monitoring of the response to TB treatment
    (Stellenbosch : Stellenbosch University, 2020, 2020-03) Mutavhatsindi, Hygon; Chegou, Novel N. ; Walzl, Gerhard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Background The accurate and rapid diagnosis of TB disease remains one of the major challenges in the control of the disease. This is mainly due to limitations in the currently existing diagnostic tests. There is also an urgent need for the development of tools that may assist in monitoring the response of TB patients to treatment. This is important given the increasing burden of multi-drug resistant TB, the risk for treatment failure and of relapse after initial clinical cure and the need for early diagnosis of such poor outcomes. The work reported in this thesis investigated different biomarkers that may assist in addressing these challenges. Aims 1) To refine and validate different biomarkers selected from literature and evaluate novel biomarker combinations in a large Africa-wide study as biosignatures for the diagnosis of TB. 2) To evaluate combinations between different biomarkers selected from the literature and mycobacterial and clinical parameters (body mass index) as tools for monitoring the response to TB treatment and prediction of relapse. 3) To employ an unbiased proteomic approach in the search for new salivary biomarkers for TB. Methods Serum and saliva specimens collected through different multicentred projects including the African-European Tuberculosis Consortium (AE-TBC), the “Action TB” study and the “Tuberculosis Research Unit” (TBRU) study at different time points were evaluated. The concentrations of host inflammatory biomarkers in serum samples from all participants (aims 1 and 2 of the project) were evaluated using the Luminex multiplex platform. The investigation of new salivary biomarkers for TB was done following a discovery proteomics approach by mass spectrometry, using the Orbitrap QExacutive platform. Standard statistical analysis procedures as used in previously published studies were employed for data analysis. Main Findings 19 out of the 20 host inflammatory biomarkers that were selected from the literature on the basis of their performance in previous studies, and evaluated as diagnostic tools for TB in the AE-TBC cohort (n=1003 study participants, 277 of whom were finally diagnosed with TB and 726 of whom were diagnosed with other respiratory diseases [ORD]), showed potential in discriminating participants with TB and those with ORD individually, thereby confirming their potential as candidate TB diagnostic biomarkers. The diagnostic potential of modified versions of a previously published 7-marker signature (Apo A1, CFH, CRP, IFN-g, IP-10, SAA and transthyretin) was confirmed. However, we identified a novel 4 marker biosignature (I- 309, CRP, IP-10, and NCAM) and a novel smaller 3-protein biosignature (CRP, I-309 and NCAM) with strong potential as diagnostic biosignatures for active TB (AUCs of 0.91 and 0.90 respectively), regardless of HIV status. When the utility of host biomarkers selected from the literature was investigated for their potential to be used as biomarkers for monitoring of the response to TB treatment, it was observed that the concentrations of multiple biomarkers changed in the course of TB treatment. The levels of SAP, IP-10, and sIL-6R amongst other biomarkers increased with treatment, whereas those of Apo CIII, MMP-2 and Fas amongst others generally tended to decrease. Some of these proteins including IL-6, IP-10, IL-22 and C3 showed potential as individual biomarkers in predicting relapse both in the action tb and TBRU cohorts. However, the most important findings from this part of the work were the identification of a combination of TTP, BMI, sICAM, IL-22, IL-1β and C3 which predicted relapse at baseline, prior to the onset of TB treatment with 89% sensitivity and 94% specificity, and a combination of Apo CIII, IP-10 and sIL-6R which predicted relapse at the end of treatment with 71% sensitivity and 74% specificity. For the unbiased proteomic identification of salivary biomarkers part of the project, we identified a total of 948 unique proteins in saliva. After statistical analysis to identify the potentially most useful proteins, we identified 26 proteins with TB diagnostic potential. Importantly, two protein biosignatures including a five-protein signature comprising of P01011, Q8NCW5, P28072, Q99574 and A0A2Q2TTZ9 and a four-protein biosignature comprising of P30043, P35579, Q99574 and P31949. The five-protein signature ascertained TB with a sensitivity of 100% and specificity of 90.91% after leave-one-out cross-validation. Conclusion The usefulness of previously identified biomarkers was confirmed in large Africa-wide diagnostic study in the current thesis. Importantly, new smaller biosignatures comprising of as little as three proteins showed strong potential in TB diagnosis. Biosignatures comprising of combinations between immunological biomarkers, BMI and TTP were identified with strong potential for use as tools for monitoring of the response to TB treatment and prediction of TB relapse both prior to the initiation of treatment, early on after initiation of treatment and at the end of treatment. Results from this thesis show that the development of a rapid point-of-care diagnostic test comprising of 3 biomarkers is possible and that such a test will potentially be highly useful if used as a triage test for TB in peripheral level health centres all over Africa, which is where our study participants were recruited. We identified strong candidate biosignatures for prediction of relapse prior to the initiation of TB treatment, early on in treatment and at the end of treatment, and also novel salivary protein biomarkers with potential in the diagnosis of TB. However, the sample sizes were relatively small, and these novel findings require validation in larger studies, especially studies conducted in multiple geographical settings.
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    Potential of host serum protein biomarkers in the diagnosis of tuberculous meningitis in children
    (Frontiers Media, 2019) Manyelo, Charles M.; Solomons, Regan; Snyders, Candice I.; Mutavhatsindi, Hygon; Manngo, Portia M.; Stanley, Kim; Walzl, Gerhard; Chegou, Novel N.
    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.
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    Prospective evaluation of host biomarkers other than interferon gamma in QuantiFERON Plus supernatants as candidates for the diagnosis of tuberculosis in symptomatic individuals
    (Elsevier, 2019-07-15) Manngo, Portia M.; Gutschmidt, Andrea; Snyders, Candice I.; Mutavhatsindi, Hygon; Manyelo, Charles M.; Makhoba, Nonjabulo S.; Ahlers, Petri; Hiemstra, Andriette; Stanley, Kim; McAnda, Shirley; Kidd, Martin; Malherbe, Stephanus T.; Walzl, Gerhard; Chegou, Novel N.
    Background: 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.