Chemokine and cytokine measurements using Luminex and Selected Reaction Monitoring (SRM): comparing technologies

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dc.contributor.advisorChegou, Novel N.en_ZA
dc.contributor.authorNkonyane, Tshepiso Ruthen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.en_ZA
dc.date.accessioned2020-11-03T12:56:36Zen_ZA
dc.date.accessioned2021-02-01T07:54:45Zen_ZA
dc.date.available2020-11-03T12:56:36Zen_ZA
dc.date.available2021-02-01T07:54:45Zen_ZA
dc.date.issued2020-12en_ZA
dc.descriptionThesis (MSc)--Stellenbosch University, 2020.en_ZA
dc.description.abstractENGLISH ABSTRACT: Background: There is an urgent need for tools for the rapid and accurate diagnosis of tuberculosis (TB) disease in resource-constrained settings. Tests based on host immunological biomarkers may be useful, especially if based on easily available samples. Objectives: To evaluate the usefulness of plasma proteins as biomarkers for diagnosing TB, to validate previously identified biosignatures [4-marker biosignature (CCL1+CRP+IP10+NCAM), 2-marker biosignature (CCL1+CRP) and 3-marker biosignatures; [(CCL1+NCAM+SAA), (CRP+IP-10+SAA), (CCL1+CRP+NCAM), (CCL1+CRP+SAP), (CRP+IP-10+MIG) and (CRP+SAP+MIG)] in a new cohort of adults suspected of having TB, and to develop a Selected Reaction Monitoring (SRM) for detecting TB protein biomarkers. Methods and Materials: We collected plasma samples from 151 individuals that presented with symptoms requiring investigation for TB disease at a primary health care clinic in the outskirts of Cape Town, South Africa, prior to assessment for TB disease. We evaluated the concentrations of 18 host markers in stored plasma samples using a multiplex platform. Using a combination of clinical, radiological and laboratory diagnosis, patients were later classified as having TB disease or other respiratory diseases (ORD). The diagnostic potential of individual analytes was analyzed using the receiver operating characteristic curve while the predictive abilities of a combination of analytes for TB disease were analyzed using the general discriminant analysis. Selected Reaction Monitoring (SRM) was used to develop an assay for the diagnosis of TB. Skyline software was used for the development of the SRM assay and analysis of the generated data. Results: Out of the 151 individuals that enrolled, 56 were pulmonary TB cases. Out of the 18 host markers that were evaluated in Chapter 3, there were significant differences in the levels of 9 host markers between patients with TB disease and those with ORDs. The concentrations of VEGF-A, IP-10, MMP-3, ferritin, SAA, CFH and CCL 1 were significantly higher in the TB cases than individuals with ORD, whereas NCAM and Apo A1 levels were significantly higher in the ORD group. When the diagnostic accuracies of individual host markers were investigated by ROC curve analysis, two markers (IP-10 and CCL 1) showed strong potential with AUC ≥0.85. When the data obtained from all study participants were fitted into GDA models regardless of HIV status, combinations between up to seven different host markers showed potential in the diagnosis of TB disease. However, an optimal diagnostic biosignature comprising of five proteins (transthyretin+MMP-3+IP-10+CFH+CCL1) diagnosed TB with an AUC of 0.90 (0.83- 0.97). Eight (8) previously identified biosignatures were assessed in study participants regardless of HIV status. A 4-marker biosignature (CCL1+CRP+IP-10+NCAM), 2-marker biosignature (CCL1+CRP) and 3-marker biosignatures [(CCL1+NCAM+SAA), (CRP+IP10+SAA), (CCL1+CRP+NCAM), (CCL1+CRP+SAP) and (CRP+IP-10+MIG)] all diagnosed TB with AUC ≥0.85, while one 3-marker signature (CRP+SAP+MIG) only diagnosed TB with AUC of 0.62. The most optimal diagnostic biosignature in all participants regardless of HIV status was the 4-marker (CCL1+CRP+IP-10+NCAM) signature which diagnosed TB with a sensitivity of 0.76(0.50-0.92) and specificity of 0.85(0.65-0.95). We further evaluated these biosignatures in HIV negative participants only and these biosignatures showed promise with areas under the ROC curves (AUC) ≥0.85 for all, except one 3-marker (CRP+SAP+MIG) biosignature. The most optimal diagnostic biosignature in HIV negative participants only was the 3-marker biosignature (CCL1+NCAM+SAA) that diagnosed TB with a sensitivity of 0.79(0.49-0.94) and specificity of 0.84(0.63-0.95). An SRM assay consisting of 83 proteins was developed, based on cytokines and chemokines that were previously investigated in diagnosing TB and reported in chapter 4. Conclusion We have shown that different candidate plasma biosignatures have potential in the diagnosis of TB disease. The observed results require further validation in a larger study. The previously identified biosignatures showed promise when validated in the current study. The biosignatures both identified and validated in this thesis hold promise as good candidate biomarkers/ biosignatures to be considered for the future development of point of-care TB tests. The developed SRM- based assay may be a useful alternative tool to diagnose TB. The assay requires further validation and optimization.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Agtergrond: Daar is 'n dringende behoefte aan instrumente vir die vinnige en akkurate diagnose van tuberkulose (TB) in hulpbronbeperkte omgewings. Toetse gebaseer op gasheerimmunologiese biomerkers kan nuttig wees, veral as dit gebaseer is op maklik beskikbare monsters. Doelwitte: Om die bruikbaarheid van plasmaproteïene as biomerkers vir die diagnosering van TB te evalueer, om voorheen geïdentifiseerde bio-handtekeninge [4-merker bio-handtekening (CCL1 + CRP + IP-10 + NCAM), 2-merker bio-handtekening (CCL1 + CRP) en 3-merker bio-handtekeninge); [(CCL1 + NCAM + SAA), (CRP + IP-10 + SAA), (CCL1 + CRP + NCAM), (CCL1 + CRP + SAP), (CRP + IP-10 + MIG) en (CRP + SAP + MIG)] in 'n nuwe groep volwassenes wat vermoedelik TB het, en om 'n geselekteerde reaksiemonitering (SRM) te ontwikkel vir die opsporing van bio-proteïene van TB-proteïene. Metodes en materiale: Ons het plasmamonsters versamel van 152 individue met simptome wat ondersoek moet word na TB-siekte in 'n primêre gesondheidsorgkliniek in die buitewyke van Kaapstad, Suid-Afrika, voor die beoordeling van TB-siektes. Ons het die konsentrasies van 18 gasheermerkers in gestoorde plasmamonsters met behulp van 'n multiplex-platform geëvalueer. Met behulp van 'n kombinasie van kliniese, radiologiese en laboratoriumdiagnose, is pasiënte later geklassifiseer as TB-siektes of ander respiratoriese siektes (ORD). Die diagnostiese potensiaal van individuele analiete is geanaliseer met behulp van die kenmerkerkromme van die ontvanger, terwyl die voorspellingsvermoë van 'n kombinasie van analiete vir TB-siekte met behulp van die algemene diskriminantanalise ontleed is. Selected Reaction Monitoring (SRM) is gebruik om 'n toets vir die diagnose van TB te ontwikkel. Skyline-sagteware is gebruik vir die ontwikkeling van die SRM-toetsing en ontleding van die gegenereerde data. Resultate: Van die 151 individue wat ingeskryf het, was 56 gevalle van long TB. Uit die 18 gasheermerkers wat in Hoofstuk 3 geëvalueer is, was daar beduidende verskille in die vlakke van 9 gasheermerkers tussen pasiënte met TB-siekte en dié met ORD's. Die konsentrasies van VEGF-A, IP-10, MMP-3, ferritin, SAA, CFH en CCL 1 was aansienlik hoër in die TB-gevalle as individue met ORD, terwyl die NCAM- en Apo A1-vlakke beduidend hoër was in die ORD-groep. Toe die diagnostiese akkuraatheid van individuele gasheermerkers deur ROC-kurwe-analise ondersoek is, het twee merkers (IP-10 en CCL 1) 'n sterk potensiaal met AUC ≥0,85 getoon. Toe die data wat van alle deelnemers aan die studie verkry is, toegepas is in GDA-modelle, ongeag MIV-status, het kombinasies van tot sewe verskillende gasheermerkers potensiaal getoon in die diagnose van TBsiekte. 'N Optimale diagnostiese biosignature wat bestaan uit vyf proteïene (transthyretin + MMP-3 + IP-10 + CFH + CCL1), het egter TB met 'n AUC van 0.90 (0.83-0.97) gediagnoseer. Agt (8) voorheen geïdentifiseerde biohandtekeninge is by die deelnemers aan die studie beoordeel, ongeag die MIV-status. 'N 4-merker bio-handtekening (CCL1 + CRP + IP-10 + NCAM), 2-merker bio-handtekening (CCL1 + CRP) en 3-merker bio-handtekeninge [(CCL1 + NCAM + SAA), (CRP + IP-10 + SAA), (CCL1 + CRP + NCAM), (CCL1 + CRP + SAP) en (CRP + IP-10 + MIG)] almal gediagnoseerde TB met AUC ≥0,85, terwyl een 3-merkteken (CRP + SAP + MIG) slegs gediagnoseer is met TB met AUC van 0,62. Die optimale diagnostiese bio-handtekening by alle deelnemers, ongeag MIV-status, was die 4-merker (CCL1 + CRP + IP-10 + NCAM) wat TB met 'n sensitiwiteit van 0,76 (0,50-0,92) en die spesifisiteit van 0,85 (0,65-0,95) gediagnoseer het. ). Ons het hierdie biohandtekeninge slegs by MIV-negatiewe deelnemers geëvalueer en hierdie biohandtekeninge het 'n belofte getoon met gebiede onder die ROC-kurwes (AUC) ≥0,85 vir almal, behalwe 'n 3-merker (CRP + SAP + MIG) bio-handtekening. Die mees optimale diagnostiese bio-handtekening by MIV-negatiewe deelnemers was slegs die 3-merker bio-handtekening (CCL1 + NCAM + SAA) wat TB gediagnoseer het met 'n sensitiwiteit van 0,79 (0,49-0,94) en spesifisiteit van 0,84 (0,63-0,95). 'N SRM-toets wat bestaan uit proteïene is ontwikkel, gebaseer op sitokiene en chemokiene wat voorheen ondersoek is in die diagnose van TB en wat in hoofstuk 4 gerapporteer is. Afsluiting: Ons het getoon dat verskillende kandidaatplasmabio-handtekeninge potensiaal het in die diagnose van TB-siekte. Die waargenome resultate vereis verdere bekragtiging in 'n groter studie. Die voorheen geïdentifiseerde biohandtekeninge het belofte getoon as dit in die huidige studie gevalideer is. Die bio-handtekeninge wat beide in hierdie proefskrif geïdentifiseer en bekragtig is, is 'n belofte vir goeie biomerkers / bio-handtekeninge wat oorweeg moet word vir die toekomstige ontwikkeling van TB-toetse wat sorgvuldig is. Die ontwikkelde SRM-gebaseerde toets kan 'n nuttige alternatiewe instrument wees om TB te diagnoseer. Die toetsing vereis verdere validering en optimalisering.en_ZA
dc.description.versionMastersen_ZA
dc.embargo.terms2023-01-01en_ZA
dc.format.extentxvi, 120 pages : illustrationsen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/109428en_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subjectchemokineen_ZA
dc.subjectcytokineen_ZA
dc.subjectLuminexen_ZA
dc.subject.lcshTuberculosis -- Diagnosis -- Cape Town (South Africa)en_ZA
dc.subject.lcshBiochemical markers -- Cape Town (South Africa)en_ZA
dc.subject.lcshBlood proteins -- Cape Town (South Africa)en_ZA
dc.subject.lcshChemokinesen_ZA
dc.subject.lcshCytokines -- Therapeutic useen_ZA
dc.subject.lcshUCTDen_ZA
dc.titleChemokine and cytokine measurements using Luminex and Selected Reaction Monitoring (SRM): comparing technologiesen_ZA
dc.typeThesisen_ZA
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