Evaluation of novel host markers detected in plasma and saliva as biosignatures for the rapid diagnosis of TB disease and monitoring of the response to TB treatment

Jacobs, Ruschca (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH SUMMARY: BACKGROUND: There is an urgent need for new tools for the rapid diagnosis of tuberculosis (TB) disease and monitoring of the response to treatment. OBJECTIVES: To investigate the usefulness of host markers detected in plasma and saliva, as well as antibodies against M. tuberculosis (M.tb) antigens, as biomarkers for the diagnosis of TB disease and monitoring of the response to treatment. To investigate the usefulness of a diagnostic approach involving the combination of antibodies and cytokines as a tool for diagnosing TB disease. METHODS: We prospectively collected plasma and saliva samples from individuals that presented with symptoms requiring investigation for 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 diseases (ORD), using a combination of clinical, radiological and laboratory findings. The concentrations of host inflammatory biomarkers were investigated in plasma and saliva samples from all study participants using a multiplex platform, whereas antibody responses against seven M.tb antigens, were investigated by ELISA. The diagnostic accuracies of individual biomarkers were assessed by receiver operator characteristics (ROC) curve analysis, whereas the accuracies of combinations between different biomarkers were assessed by General Discriminant Analysis (GDA). RESULTS: Of the 74 host markers evaluated in plasma, 18 showed diagnostic potential as determined by area under the ROC curve (AUC), with the most promising being NCAM, CRP, SAP, IP-10, ferritin, TPA, I-309, and MIG, which diagnosed TB disease individually with AUC ≥0.80. A six-marker plasma protein biosignature comprising of NCAM, SAP, IL-1β, sCD40L, IL-13 and Apo A-1 diagnosed TB disease with a sensitivity of 100% (95% CI, 86.3-100%) and specificity of 89.3% (95% CI, 67.6-97.3%), irrespective of HIV status, whereas six-marker plasma protein biosignatures diagnosed TB disease with 100% accuracy in the absence of HIV. Of the 69 host markers that were investigated in saliva, only two (IL-16 and IL-23) showed diagnostic potential with AUC ≥0.70. A five-marker salivary 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%), regardless of HIV infection status, whereas eight-marker salivary 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. IgA responses against four M.tb antigens (NarL, Rv3019c, “Kit1” and “Kit2”) were significantly different between TB patients and individuals with ORD, with combinations between different antibodies diagnosing TB disease with an AUC of 0.80. The diagnostic accuracy of the antibodies increased when used in combination with patient’s symptoms or cytokines. Finally, the concentrations of biomarkers detected in plasma and saliva changed during TB treatment, thereby indicating that they may be useful in monitoring of the response to TB treatment. CONCLUSIONS: We have identified novel plasma and 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.

AFRIKAANSE OPSOMMING: AGTERGROND Daar is 'n dringende behoefte aan nuwe toestelle vir die vinnige diagnose van tuberkulose ( TB ) en monitering van die reaksie op behandeling. DOELWITTE Om die nut van gasheer merkers in plasma en speeksel waar te neem , sowel as teenliggaampies teen M. tuberculosis ( M.tb ) antigene , as biomerkers vir die diagnose van TB en monitering van die reaksie op behandeling te ondersoek . Om die nut van 'n diagnostiese benadering met betrekking tot die kombinasie van teenliggaampies en sitokiene as 'n toestel vir die diagnose van TB te ondersoek. METODES Ons het plasma en speeksel monsters van individue met simptome wat tot die ondersoek van TB dui vooruitwerkend ingesamel by 'n gesondheidsentrum in Kaapstad , Suid-Afrika , voor die vestiging van 'n kliniese diagnose. Pasiënte was later geklassifiseer as TB of ander respiratoriese siektes (ARD) pasiënte, met behulp van 'n kombinasie van kliniese , radiologiese en laboratorium bevindings. Die konsentrasies van die gasheer inflammatoriese biomerkers in al die studie deelnemers in plasma en speeksel monsters was ondersoek met behulp van 'n multiplex platform , terwyl teenliggaam response teen sewe M.tb antigene , ondersoek was met ELISA . Die diagnostiese akkuraatheid van individuele biomerkers is beoordeel deur ontvanger operateur eienskappe (OOC) kurwe analise ,terwyl die akkuraatheid van kombinasies tussen verskillende biomerkers beoordeel was deur Algemene Diskriminant Analise ( GDA ) . RESULTATE Van die 74 gasheer merkers wat geëvalueer was in plasma het 18 diagnostiese potensiaal gehad soos bepaal deur area onder die OOC kurwe (AOC), met NCAM, CRP, SAP, IP-10, Ferritin, TPA, I-309, en MIG as die mees belowende merkers wat TB individueel diagnoseer met AOC ≥0.80. 'n Ses-merker plasmaproteïen biosignature bestaande uit NCAM, SAP, IL-1β, sCD40L, IL-13 en Apo A-1 het TB gediagnoseer met 'n sensitiwiteit van 100% (95% CI, 86,3-100%) en spesifisiteit van 89,3 % (95% CI, 67,6-97,3%), ongeag MIV-status, terwyl ‘n ses-merker plasmaproteïen biosignatures TB gediagnoseer het met 100% akkuraatheid in die afwesigheid van MIV. Van die 69 gasheer merkers wat ondersoek was in speeksel het slegs twee (IL-16 en IL-23) diagnostiese potensiaal getoon met AOC ≥0.70. 'n Vyf-merker speeksel biosignature bestaande uit IL-1β, IL-23, ECM-1, HCC1 en fibrinogeen het TB gediagnoseer met 'n sensitiwiteit van 88,9% (95% CI, 76,7-99,9%) en spesifisiteit van 89,7% (95% GI, 60,4-96,6%), ongeag van MIV-infeksie status, terwyl agt-merker speeksel biosignatures gegenereer was met 'n sensitiwiteit van 100% (95% CI, 83,2-100%) en spesifisiteit van 95% (95% CI, 68,1-99,9 %) in die afwesigheid van MIV-infeksie. IgA reaksies teen vier M.tb antigene (NarL, Rv3019c, "Kit1" en "Kit2") het aansienlik verskil tussen TB-pasiënte en individue met ARD, met kombinasies tussen verskillende teenliggaampies wat TB diagnoseer met 'n AOC van 0.80. Die diagnostiese akkuraatheid van die teenliggaampies verhoog wanneer dit gebruik word in kombinasie met pasiënt simptome of sitokiene. Ten slotte, die konsentrasies van biomerkers wat in plasma en speeksel ondersoek was verander tydens TB behandeling, en dui sodoende aan dat hulle nuttig kan wees in die monitering van die reaksie op TB behandeling. GEVOLGTREKKING Ons het nuwe plasma en speeksel biosignatures geïdentifiseer wat nuttig kan wees in die diagnose van TB en monitering van die reaksie op TB behandeling. Ons bevindinge vereis verdere bekragtiging in groter studies.

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