Innovative strategies to improve the diagnosis of intrathoracic tuberculosis in children

Walters, Elisabetta (2018-12)

Thesis (PhD)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Paediatric tuberculosis (TB) contributes approximately 10% of the global TB burden, with over one million estimated new cases and 253,000 TB-related deaths in children during 2016. Paediatric TB is a particular problem in low and middle income countries. However, the majority of paediatric cases were not notified to National TB Programs or the World Health Organization and >96% of deaths were estimated to have occurred in children who were not receiving antituberculosis treatment. Young, HIVinfected and malnourished children progress rapidly from infection with Mycobacterium tuberculosis (M.tb) to TB, and are at exquisite risk of significant morbidity and mortality from complicated and disseminated forms of TB. The challenges around the diagnosis and microbiological confirmation of pulmonary TB (PTB), the most common manifestation of TB disease in children, contribute to poor access to appropriate treatment and to underreporting. The diagnosis of TB in young children typically relies on the evaluation of clinical symptoms and epidemiological factors, and, if available, includes tests of TB infection and chest radiology. All of these tools have considerable limitations and cannot reliably confirm or exclude a diagnosis of PTB. However, the bacteriological confirmation of PTB in children requires the collection of respiratory specimens using procedures that are both relatively invasive and resource-intensive. Furthermore, the current gold standard of diagnosis, mycobacterial culture, has low sensitivity (approximately 30%) and long turnaround time (up to 6 weeks) in children, who typically have paucibacillary TB (low bacillary load). In resource-limited settings, the capacity for respiratory sampling of young children is typically low. These diagnostic challenges prevent adequate reporting and global surveillance of paediatric TB. Diagnostic uncertainty also compromises the clinical management of paediatric PTB, resulting in over- and under-treatment, and has resulted in the systematic exclusion of children from much-needed interventional research, including tuberculosis treatment trials. Diagnostic research in paediatric PTB has also been poorly standardised, making generalizability and comparability of results difficult. In addition, the insensitive reference standard has hindered progress towards the development of new diagnostic tests tailored for children. In an effort to develop and investigate more feasible strategies to improve and promote microbiological testing of children with suspected PTB living in high TB-burden settings, I enrolled a large well-characterized cohort of children presenting to hospital with suspected PTB. Children were thoroughly investigated, using standard approaches and intensive specimen collection for liquid culture and molecular testing by Xpert ® MTB/RIF (Xpert). Chest radiographs were dual read by blinded experts and reported using standard forms. All children were followed regardless of their final diagnosis and the spectrum of TB and non-TB disease was well described. I evaluated a number of novel diagnostic strategies, including the use of stool specimens for diagnosis of PTB using culture and Xpert, using different stool processing methods, and pooling respiratory specimens to improve the diagnostic yield and reduce the cost of laboratory testing. Importantly, I developed a framework for future evaluation of novel diagnostic tools/ biomarkers for the diagnosis of PTB in children. The total cohort included 608 children and was representative of the demographics and spectrum of disease observed in many high TB-burden settings, where young children bear the highest burden of TB disease. The median age of the cohort was 16.2 months, with 11.8% HIV-infected. Infants below 6 months of age constituted almost 15% of the total cohort. More than 20% of children had a non-specific clinical presentation, with similar prevalence of acute respiratory symptoms across all age groups and diagnostic categories. Radiological features not typically associated with PTB were common, and indicate a high burden of respiratory pathology as well as potentially non-typical radiological manifestations of PTB. Two hundred and eighty-one (46.2%) children were diagnosed with PTB and were prescribed antituberculosis treatment: 117 (41.6%) were microbiologically confirmed by Xpert or culture, which represents a high diagnostic yield, considering that approximately 50% of children with PTB had nonsevere pulmonary disease. In addition, 20/327 (6.6%) children initially considered symptomatic controls were initiated on antituberculosis treatment within two months of enrolment, due to poor clinical progress or positive results from baseline and follow-up bacteriological investigations. This emphasizes the importance and utility of careful specimen collection, incorporating different specimen types and different diagnostic tests and of follow-up of all children in whom there is a clinical suspicion of PTB. An unexpectedly high proportion of young infants <6 months of age had severe PTB, including cavities, associated with high bacillary load and smear-positivity. In addition, young infants and HIV-infected children were high-risk groups for disseminated TB. This calls for urgent priority to be given towards the development of tailored diagnostic tests that can rapidly confirm and quantify M.tb disease in the youngest children, and in the early stages of disease, prior to rapid progression to severe TB. Careful consideration should be given to infection control measures when managing and investigating children, including young infants with suspected PTB. I showed that stool as a specimen was useful to confirm M.tb using Xpert in children with severe pulmonary disease, particularly in children with cavities on chest radiograph, detecting 45% of those who were bacteriologically confirmed on respiratory specimens. A novel centrifugation-free processing method for stool specimens (stool processing kit) showed similar results to the more laborious, centrifugation-dependent methods I initially investigated. This new approach could be used with more sensitive molecular assays in future to improve stool-based diagnosis of PTB in children. In contrast, stool culture had limited value in the detection of M.tb, primarily due to very high contamination (>41% of stool cultures) using standard N-acetyl-l-cysteine–sodium hydroxide (NALC-NaOH 1.25%) decontamination protocols. Finally, I showed that pooling up to three respiratory specimens of different types (gastric aspirate, induced sputum and nasopharyngeal aspirate) per child, in children who could not expectorate sputum, had similar diagnostic yield by Xpert and culture as individually testing the same three single respiratory specimens. In paired analyses, pooled specimens had significantly higher overall yield than induced sputum and nasopharyngeal aspirate alone, but had similar diagnostic yield as a single gastric aspirate (86.5% vs. 74.4% respectively, p=0.46). The overall yield of three individual specimens tested individually was 86% of all confirmed cases, similar to the overall yield of pooled specimens. These results support the substantial diagnostic value of a single gastric aspirate using culture and Xpert, and of “front-loading” specimens of different types on one day to improve the feasibility of specimen collection in young children. Through this cohort study, I collected comprehensive follow-up data documenting response to antituberculosis treatment and clinical progress in children not receiving antituberculosis treatment (symptomatic controls), to 6 months. These data will be further analysed to validate recently proposed clinical case definitions for TB diagnostic research in children, including the diagnostic value of clinical and other follow-up measures. Symptomatic controls who initially presented with symptoms suggestive of PTB will be further analysed to better understand the spectrum of non-TB respiratory disease borne by children from high-TB burden settings. I have also established a bio-repository of well-characterised blood and urine specimens for evaluation of promising diagnostic and prognostic biomarkers of TB disease in children. In summary, through this body of research, I have generated novel data on the utility of several feasible diagnostic strategies for the diagnosis of PTB in HIV-infected and uninfected children from high TB-burden settings. I have analysed these data in relation to relevant clinical and laboratory characteristics in order to make specific recommendations on the most appropriate placement of these strategies, considering both target populations and different levels of health care. I was able to do this by carrying out a well-designed study, in a well-described cohort and by comprehensively reporting on all aspects of the study, including non-evaluable results and complex clinical scenarios. These aspects should be considered when future diagnostic studies for paediatric PTB are being designed, implemented and reported. I have created a rigorous framework for the evaluation of future novel diagnostic strategies, and I have identified numerous areas which require further research and intervention.

AFRIKAANSE OPSOMMING: Tuberkulose (TB) in kinders dra by tot ongeveer 10% van die globale las van TB ter wêreld, met ongeveer 1 miljoen nuwe gevalle en ‘n geskatte 253,000 TB-verwante sterftes in kinders in 2016. Tuberkulose in kinders is hoofsaaklik ń probleem in middleen lae-inkomste lande. Die meerderheid van kindertuberkulose gevalle word egter nie aangemeld aan Nasionale Tuberkulose Kontroleprogramme, of die Wêreld Gesondheid Organisie (WGO) nie, en meer as 96% van die geskatte TB sterftes onder kinders was in kinders wat nie antituberkulose behandeling ontvang het nie. In jong kinders, MIVgeïnfekteerde en wangevoede kinders gaan TB vinning oor vanaf infeksie met Mycobacterium tuberculosis (M.tb) na TB siekte, en hierdie kinders het ‘n besondere hoë risiko vir betekenisvolle morbideit en mortaliteit as gevolg van gekompliseerde en gedissemineerde vorms van TB. Die uitdagings rondom die diagnose en mikrobiologiese bevestiging van pulmonale TB (PTB), die mees algemene vorm van TB in kinders, dra verder by tot die wêreldwye swak toegang tot toepaslike behandeling en die onderberiggewing van tuberkulose in kinders. Die diagnose van PTB in jong kinders maak tipies staat op die evaluering van kliniese simptome en epidemiologiese faktore, en, indien beskikbaar, sluit dit ook toetse van TB infeksie en borskasplate in. Al hierdie hulpmiddels het aansienlike beperkinge en kan nie betroubaar die diagnose van TB bevestig of uitsluit nie. Die bakteriologiese bevestiging van PTB in kinders vereis egter die versameling van respiratoriese monsters, wat beide relatief indringend en hulpbron-intensief is. Verder het die huidige goudstandaard van TB diagnose, naamlik kultuur van Mycobacterium tuberculosis, ń lae sensitiwiteit (ongeveer 30%) en het ‘n lang omkeertyd (tot 6 weke) in kinders wat TB kry, met ń lae hoeveelheid Mycobacterium organismes. In omgewings met min hulpbronne, is die kapasiteit om respiratoriese monsters in jong kinders te versamel, tipies laag. Hierdie diagnostiese uitdagings voorkom dat daar toepaslike verslaggewing en globale waarneming is oor kindertuberkulose is. Hierdie diagnostiese onsekerheid ondermyn ook die kliniese sorg van PTB in kinders, wat tot oor-en onderbehandeling lei. Tot dusver het dit ook gelei tot die sistematiese uitsluiting van kinders in broodnodige intervensionele navorsing, insluitend tuberkulose-middelproewe. Diagnostiese navorsing in PTB in kinders was tot dusver ook swak gestandardiseer, wat veralgemening en vergelyking van verskillende studiebevindinge moeilik maak. Addisioneel het die onsensitiewe diagnostiese verwysingsstanddaard vooruitgang om nuwe diagnostiese toetse vir kindertuberkulose te ontwikkel, belemmer. In ‘n poging om meer haalbare strategieë te ontwikkel om die mikrobiologiese toetsing te verbeter en te bevorder in kinders met vermoedelike PTB wat in areas woon met ‘n hoë TB-las, het ek ‘n goed-gekarakteriseerde kohort van kinders wat voorgedoen het met vermoedelike PTB by hospitale, ingesluit in ‘n studie. Kinders is deeglik ondersoek, met die gebruik van gestandardiseerde metodes en met intensiewe monsterversameling vir vloeibare kultuur en molekulêre toetsing met Xpert® MTB/RIF (Xpert). Borkasplate is dubbel gelees deur kliniese kenners en gerapporteer op standaard vorms. Alle kinders is opgevolg, onafhanklik van hulle finale diagnose en hul spektrum van TB siekte; kinders met nie-TB verwante siekte is ook goed beskryf. Ek het ‘n aantal nuwe diagnostiese strategieë evalueer, insluitende die gebruik van stoelgangmonsters vir die diagnose van PTB met kultuur en Xpert, asook die gebruik van verskillende stoelgangprosesseringsmetodes. Ek het die waarde van gekombineerde respiratoriese monsters evalueer in ‘n poging om die diagnostiese opbrengs te verbeter, en om labaratoriumkostes te verminder. Van belang is dat ek ‘n konseptuele en kliniese raamwerk ontwikkel het vir die toekomstige evaluering van nuwe diagnostiese toetse en biomerkers vir die diagnose van PTB in kinders. Die totale kohort het 608 kinders ingesluit en was verteenwoordigend van die demografie en spektrum van TB in kinders wat in baie hoë-las areas gesien word, waar die meeste kindertuberkulose in jong kinders voorkom. Die mediane ouderdom van die kinders in die kohort was 16.2 maande, met 11.8% van kinders MIV-geïnfekteer. Babas onder 6 maande ouderdom het amper 15% van die kohort uitgemaak. Meer as 20% van kinders het met atipiese kliniese simptome voorgedoen, met ‘n soortgelyke prevalensie van akute simptome in all ouderdomsgroepe en diagnostiese kategorieë. Radiologiese kenmerke wat nie tipies van PTB is nie, was algemeen, en dui op die hoë las van respiratoriese patologie, asook die potensiële atipiese radiologiese voordoening van PTB. Twee-honderd-een-en-tagtig (46.2%) kinders is gediagnoseer met PTB en aan hulle is antituberkulose-behanding voorgeskryf: 117 (41.6%) is bakteriologies bevestig met Xpert of kultuur, ‘n hoē diagnostiese opbrengs, as mens in aanmerking neem dat ongeveer 50% van kinders met PTB, nie erge vorms van PTB gehad het nie. Verder is 20/327 (6.6%) kinders wat aanvanklik as simptomatiese kontroles geklassifiseer was, op antituberkulose behandeling begin, binne twee maande nadat hulle in die studie ingesluit is, as gevolg van swak kliniese respons of positiewe bakteriologiese toetsing wat aanvanklik of gedurende opvolgbesoeke, gedoen is. Hierdie data beklemtoon die waarde van noukeurige monstervesameling, insluitend verskillende tipes monsters en diagnostiese toetse, en die belang van kliniese opvolg van alle kinders met vermoedelike TB. ‘n Onverwagte hoë proporsie van jong babas onder die ouderdom van 6 maande, het erge vorms van PTB gehad, insluitende kaviteite, wat geassosieer was met ‘n hoë bakterie lading en smeer-positiwiteit. Verder was jong babas en MIV-geïnfekteerde kinders hoë-risiko-groepe vir gedissemineerde TB. Hierdie data beklemtoon die dringende prioritisering wat gegee moet word aan die ontwikkeling van toepaslike diagnostiese toetse wat M.tb vinning kan diagnoseer en kwantifiseer in die vroeë stadiums van siekte, voordat TB vinnig na na erge vorms van siekte progresseer in kinders. Versigtige aandag moet ook gegee word aan infeksiebeheer met die ondersoek en hantering van TB in kinders, insluitende jong babas met vermoedelike TB. Ek het getoon dat stoelgang monsters nuttig is om M.tb te bevestig met Xpert in kinders met erge vorms van TB longsiekte, veral in kinders met longkaviteite, in wie stoelgang toetse TB bevestig het in 45% van kinders wat positiewe respiratoriese monsters gehad het. ‘n Nuwe sentrifigureer-vrye prosesseermetode vir stoelgangmonsters (stoelgang toets stel) het soortgelyke resultate getoon as die meer intensiewe sentrifiguurmetodes wat ek inisieël ondersoek het. Hierdie nuwe benadering sou gebruik kon word met meer sensitiewe molekulêre toetse in die toekoms om die bevesting van longtuberkulose in kinders deur stoelgangtoetsing, te verbeter. Aan die ander kant, was die gebruik van stoelgangkultuur van min waarde om M.tb te diagnoseer, primêr vanweë die hoë kontaminasierisiko (>41% van stoelgangkulture), met die gebruik van die huidige standaard NALC-NaOH (1.25%) dekontaminasie-protokolle. Ter slotsom, het ek ook getoon dat in kinders wat nie spontaan sputum kon uithoes nie, die toetsing van tot 3 respiratoriese monsters per kind van verskeie tipes (maagsappe, geïnduseerde sputum en nasofaringeale aspirate) in kombinasie, ‘n soortgelyke opbrengs gehad het met Xpert en kultuur, as monsters wat individueel getoets was op die drie monsters. In gepaarde analise, het gekombineerde monsters ‘n betekenisvolle hoër opbrengs gehad as geïnduseerde sputum en nasofaringeale aspirate op hul eie, maar het ‘n soortgelyke opbrengs gehad as ‘n enkele maagsap (86.5% vs. 74.4%, onderskeidelik, p=0.46). Die algehele opbrengs van die drie individiduele respiratoriese monsters wat invidiueel getoets is, was 86% van alle bevestigde gevalle, soortgelyk aan die algemene opbrengs van gekombineerde monsters. Hierdie resultate ondersteun die aansienlike diagnostiese waarde van ‘n enkele maagsap met Xpert en kultuur, en van die nut van ‘n diagnostiese “voorladingstrategie” met verskeie monsters wat op een dag versamel word, om die haalbaarheid van monsterversameling vir die diagnose van PTB in jong kinders te verbeter. Gedurende hierdie kohortstudie, het ek omvattende opvolgdata versamel wat die respons op antitberkulose-behandeling, asook die kliniese verbetering in kinders wat nooit antituberkulose behandeling ontvang het nie (simptomatiese kontroles), beskryf, tot en met 6 maande opvolg. Hierdie data sal verder analiseer word om die diagnostiese waarde van die huidige voorgestelde kliniese gevalsdefinisies te evalueer, asook die diagnostiese waarde van kliniese en ander opvolgondersoeke. Kinders wat inisieël as simptomatiese kontroles voorgedoen het met simptome verdag van PTB, sal verder analiseer word om die spektrum van non-tuberkulose-verwante longsiekte in kinders in areas met ‘n hoë las van TB te karakateriseer. Ek het ook ‘n versameling van goedgekarakteriseerde monsters insluitende blood en urine, vir die toekomstige evaluering van belowende diagnostiese en prognostiese biomerkers vir kindertuberkulose, gevestig. Ter opsomming, deur hierdie navorsing het ek nuwe data versamel oor die gebruik van nuwe toetse en diagnostiese strategieë vir die diagnose van kinderlongtuberkulose in MIV-geïnfekteerde en on geïnfekteerde kinders in hoë lastuberkulose areas. Ek het hierdie data geanaliseer met betrekking tot relevante kliniese en laboratorium-eienskappe sodat ek spesifieke aanbevelings kon maak rondom die mees toepaslike plasing van hierdie strategieë, met inagneming van beidie die teikenpopulasies en verskillende vlakke van gesondheidsorg. Ek kon dit behaal deur ‘n goed-ontwerpte studie uit te voer, in ‘n goed-gekarakteriseerde kohort en deur omvattend verslag te doen oor alle aspekte van die studie, insluitende nie-evalueerbare resultate, en komplekse kliniese scenarios. Al hierdie aspekte moet in ag geneem word met die beplanning, analise, en verslaggewing van toekomstige studies om PTB in kinders te diagnoseer. Ek het ‘n streng raamwerk gebou vir die evaluering van toekomstige nuwe diagnostiese strategieë vir PTB in kinders, en het verskeie areas identifiseer wat verdere navorsing en intervensie benodig.

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