Evaluation of the potential of Mycobacterium tuberculosis antigen-specific host biomarkers detected in QuantiFERON® TB GOLD Plus supernatants in the diagnosis of TB disease

Manngo, Makhadzi Portia (2018-12)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: BACKGROUND The diagnosis of tuberculosis (TB) disease remains a challenge. This is mainly due to limitations with the current TB diagnostic tests including unavailability of rapid pointof-care tests. New TB diagnostic tests are therefore urgently needed. The QuantiFERON-TB® Gold (QFT) Plus test is a recently introduced test for the diagnosis of M. tb infection, and disease in some patient groups. As this is a relatively new test which is currently in use worldwide, it is important that its performance be evaluated, especially in high TB burden settings. Furthermore, it is not known whether measurement of host markers other than Interferon-gamma in culture supernatants of individuals with active TB or other respiratory diseases (ORD), has potential in the diagnosis of TB disease. OBJECTIVES 1) To evaluate the usefulness of the QFT Plus test in the diagnosis of TB disease, and assess the utility of the test, when used in combination with symptoms, as a tool for diagnosis of TB disease in people suspected of having active TB in a high burden setting. 2) To evaluate alternative host biomarkers detected in QFT Plus supernatants, other than IFN-γ as biosignatures for the diagnosis of active TB METHODS We recruited 120 participants presenting at a primary health care clinics in Cape Town, South Africa with symptoms requiring investigation for TB disease. These participants formed part of a larger ongoing biomarker project known as the ‘ScreenTB’ study. Participants were later classified as TB or ORD based on the results of clinical and laboratory tests. After performing the standard QFT Plus test in study participants, the concentrations of 37 host biomarkers were evaluated in culture supernatants using a multiplex immunoassay. RESULTS Out of 120 individuals included in the study, 35 (29.2%) were diagnosed with active TB and were culture positive. The QFT Plus test diagnosed TB disease in all study participants with sensitivity and specificity >70%. A combination of symptoms including cough, fever and weight loss diagnosed TB disease with sensitivity and specificity >70% with an area under the receiver operator characteristics curve of 0.81. Multiple host biomarkers detected in the unstimulated and antigen-stimulated QFT Plus tubes showed potential as diagnostic markers for TB. Individual markers which diagnosed TB disease with sensitivities and specificities >60% included ITAC-1, IL-3, I-309, MIG, and EGF, P-selectin. Combinations between host biomarkers showed potential in the diagnosis of TB disease with a six-marker biosignature derived from unstimulated supernatants (APO-CII, ITAC-1, MIG, MCP-2, I-309, and NCAM-1) diagnosing TB disease with a sensitivity and specificity >78%, a four-marker TB1 and TB2 antigenspecific biosignature (TNFα, LIGHT, MIG and P-selectin ) which diagnosed TB disease with sensitivity and specificity >73%, after leave-one-out cross validation. CONCLUSION The sensitivity of the QFT Plus test for active TB was inferior to the published >80% mentioned in the package insert by the manufacturer. Host biomarkers detected in QFT Plus supernatants showed potential in the diagnosis of active TB disease. Further validation studies are needed before such markers may be considered as candidate biomarkers for a blood-based diagnostic test for active TB.

AFRIKAANSE OPSOMMING: AGTERGROND Die diagnose van tuberkulose (TB siekte) bly 'n uitdaging. Dit is hoofsaaklik te wyte aan beperkinge met die huidige TB diagnostiese toetse, insluitend die beskikbaarheid van vinnige punt van soegtoetse. Nuwe TB diagnostiese toetse is dus dringend nodig. Die QuantiFERON-TB® Goud (QFT) Plus toets is 'n onlangs ontwikkelde toets vir die diagnose van M. tb infeksie en tuberkulose in sommige pasiëntgroepe. Aangesien dit 'n relatief nuwe toets is wat tans wêreldwyd gebruik word, is dit belangrik dat die prestasie vd toets geëvalueer word, veral in hoë TB-lasinsweld dtrelie tellings. Verder is dit nie bekend of meting van gasheermerkers behalwe Interferon-gamma in kweek supernatante van individue met aktiewe TB of ander respiratoriese siektes (ORD), potensiaal het in die diagnose van TB-siekte. DOELWITTE 1) Om die nut van die QFT Plus-toets in die diagnose van TB-siekte te evalueer, wanneer dit in kombinasie met simptome gebruik word, as 'n instrument vir die diagnose van TB-siekte by mense wat vermoed word dat hulle aktiewe TB heit in 'n hoë lastrehe. 2) Om alternatiewe gasheerbiomerkers wat in QFT Plus supernatante aangetref word, te evalueer, anders as IFN-γ as biomeker vir die diagnose van aktiewe TB METODES Ons het 120 deelnemers gewerf by 'n primêre gesondheidsorgkliniek in Kaapstad, Suid-Afrika, met simptome wat ondersoek na TB-siekte vereis. Hierdie deelnemers het deel gevorm van 'n groter voortgesette biomerkerprojek wat bekend staan as die 'ScreenTB'-studie. Deelnemers is geklassifiseer as TB of ORD gebaseer op die resultate van kliniese en laboratoriumtoetse. Nadat die standaard QFT Plus-toets in studie-deelnemers uitgevoer is, is die konsentrasies van 37 gasheerbiomerkers geëvalueer in kweek supernatante met behulp van 'n veelvuldige imuuufoetse. RESULTATE Uit 120 individue wat in die studie ingesluit is, is 35 (29,2%) met aktiewe TB gediagnoseer en was kultuur positief. Die QFT Plus-toets het TB-siektes in alle studiedeelnemers met 'n sensitiwiteit en spesifisiteit van> 70% gediagnoseer. 'n Kombinasie van simptome soos hoes, koors en gewigsverlies diagnoseer TB siekte met sensitiwiteit en spesifisiteit> 70% met 'n gebied onder die ontvanger operateur eienskappe kurwe van 0.81. Veelvuldige gasheerbiomerkers wat in die ongestimuleerde en antigeen-gestimuleerde QFT Plus-buise opgespoor is, het potensiaal as diagnostiese merkers vir TB vertoon. Individuele merkers wat TB-siekte gediagnoseer het met sensitiwiteit en spesifieke eienskappe> 60% sluit in ITAC-1, IL3, I-309, MIG en EGF, P-selektien. Kombinasies tussen gasheerbiomarkers het potensiaal getoon in die diagnose van TB-siekte. Ses biomakers van ongestimuleerde supernatante (APO-CII, ITAC-1, MIG, MCP-2, I-309, en NCAM-1) het 'n sensitiwiteit en spesifisiteit getoon van >78%. Die vier-biomeker TB1 en TB2 antigenspesifieke kombinasie (TNFa, LIG, MIG en P-selektien) het TB slette gecliagnoseer met 'n sensitiwiteit en spesifisileit >73% na verlof-een-uit kruis validasie. AFSLUITING Die sensitiwiteit van die QFT Plus-toets vir aktiewe TB was nie soos die gepubliseerde> 80% wat in die pakketstuk deur die vervaardiger genoem word nie. Gasheer biomerkers wat in QFT Plus supernatante aangetoon is, het potensiaal getoon in die diagnose van aktiewe TB-siekte. Verdere valideringstudies is nodig voordat sulke merkers as kandidaat-biomerkers beskou kan word vir 'n bloedgebaseerde diagnostiese toets vir aktiewe TB.

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