Investigating the role of respiratory co-infections and the nasopharyngeal microbiome in children with suspected pulmonary tuberculosis

Hamman, Bianca (2020-12)

Thesis (MSc)--Stellenbosch University, 2020.

Thesis

Introduction: Tuberculosis (TB) is a global health problem, causing morbidity, mortality and devastating social and economic impacts. Pediatric TB is particularly challenging due to difficulties in diagnosis. Children are particularly susceptible to respiratory infections and this may be influenced by the microbial colonization of the respiratory tract, which may play a role in the clinical presentation and pathogenesis of TB. The nasopharyngeal microbiome is critical for respiratory health and may impact on the development, presentation and diagnosis of TB disease. Antibiotics contribute to microbial dysbiosis which may lead to the development, progression or exacerbation of other diseases. However, there is limited data describing the nasopharyngeal microbiota of children with and without TB, or the effect of TB treatment on the nasopharyngeal microbiome. Methods: Respiratory samples were obtained from pediatric patients with suspected pulmonary TB (PTB) at baseline and follow up visits (2 and 6 months). Participants were classified as having bacteriologically confirmed PTB, clinically diagnosed PTB or unlikely PTB (well-defined ill controls). Respiratory pathogens were detected in all baseline respiratory samples using the Seegene Allplex™ Respiratory Panel 4 and a Pneumocystis jirovecii real-time PCR assay. The nasopharyngeal microbiome of 26 participants was characterized and the effect of TB treatment determined by 16S rRNA sequencing, using the Illumina Miseq platform. Results: Seventy children were included; 27.1% were categorized with bacteriologically confirmed PTB, 32.9% with clinically diagnosed PTB and 40% with unlikely PTB. The most frequently detected bacterial pathogens were Haemophilus influenzae (52/70, 74.2%) and Streptococcus pneumoniae(42/70, 60%). There was no association between the presence of bacterial pathobionts/pathogens and TB disease. Due to poor sequence quality resulting from load shedding during sequencing, the reverse reads were excluded from microbiome analysis. The most commonly detected phyla in all samples were Proteobacteria, Fusobacteria, Firmicutes and Bacteroidetes. Common familia included Streptococcaceae, Pasteurellaceae, Moraxellaceae, Prevotellaceae, Veillonellaceae and Neisseriaceae. There were no significant differences in the microbiome profile or alpha and beta diversity between TB cases and controls at baseline. However, differential abundance testing showed 4-5 fold differences in abundance of Pasteurellaceae and Prevotellaceae between the TB cases and ill controls. There was also no significant difference in microbiota profile or alpha diversity at 2 or 6 months in TB cases, who received TB treatment. However, differential abundance testing identified a reduction in the abundance of Veillonellaceae, Staphylococcaceae, Prevotellaceae, Neisseriaceae, Enterobacteriaceae and Aerococcaceae in TB cases after treatment. Conclusion: This study observed no significant differences between the respiratory pathogens in children with and without PTB. Similarly, no differences in alpha or beta diversity were observed between the respiratory microbiota of TB cases and controls, or after TB treatment. However, differences in abundance of some families, between TB cases and controls at baseline, and before and after TB treatment, suggest that further research on this topic is warranted, considering the numerous limitations which may have impacted the findings of this study. This study contributed to the data available regarding respiratory microbiota in children with suspected PTB in a TB endemic setting and highlighted the challenges of conducting microbiome research in resource limited settings.

Inleiding:Tuberkulose (TB) is ‘n wêreldwye gesondheidsprobleem wat morbiditeit en mortaliteit veroorsaak en verrykende sosiale en ekonomiese impakte het. Pediatriese TB is veral uitdagend weens diagnose moeilikhede. Kinders is veral vatbaar vir respiratoriese infeksies en dit kan beïnvloed word deur die mikrobiese kolonisasie van die respiratoriese kanaal wat ‘n rol kan speel in die kliniese aanbieding en patogenese van TB. Die nasofaringeale mikrobioom is krities vir respiratoriese gesondheid en kan die ontwikkeling, aanbieding en diagnose van TB beïnvloed. Antibiotika kan bydra tot disbiose van die nasofaringeale mikrobiota wat kan lei tot die ontwikkeling, bevordering of verergering van ander siektes. Alhoewel, daar is beperkte data wat die nasofaringeale mikrobiota van kinders met en sonder TB, of die effek van TB behandeling op die nasofaringeale mikrobioom beskryf. Metodes: Respiratoriese monsters is geneem vanaf pediatriese pasiënte met moontlike pulmonêre TB by basislyn- en opvolgbesoeke (2 en 6 maande). Deelnemers is geklassifiseer as volg: met bakteriologies-bevestigde PTB, klinies gediagnoseerde PTB of onwaarskynlike PTB (goed beskryfde siek kontrole). Respiratoriese patogene is in alle basislyn respiratoriese monsters opgespoor, deur die gebruik van Seegene Allplex™ Respiratory Panel 4 en ‘n Pneumocystis jirovecii PKR toets. Die nasofaringeale mikrobioom van 26 deelneemers was karakteriseer en die effek van TB behandeling bepaal, deur 16S rRNA volgordebepaling, geteiken met Illumina Miseq tegnologie. Resultate: Sewentig kinders is ingesluit; 27.1% met bakteriologies-bevestigde PTB geklassifiseer is, 32.9% met klinies gediagnoseerde PTB en 40% met onwaarskynlike PTB. Die mees algemeen opgespoorde bakteriële patogene was Haemophilus influenzae (52/70, 74.2%) en Streptococcus pneumoniae(42/70, 60%). Daar was geen verwantskap tussen die teenwoordigheid van sekere bakteriële “pathobionts”/patogene en TB siekte nie.As gevolg van die slegte kwaliteit van die volgordes as gevolg van beurtkrag, was die “reverse reads” nie ingesluit in die mikrobioom analise nie. Proteobacteria, Fusobacteria, Firmicutes en Bacteroidetes was die mees algemeen opgespoorde filums. Algemene gesinne het onder andere Streptococcaceae, Pasteurellaceae, Moraxellaceae, Prevotellaceae, Veillonellaceae en Neisseriaceae ingesluit. Daar was geen merkwaardige verskille in die mikrobiota profiele of alfa en beta diversiteit tussen TB gevalle en kontrole by basislyn nie. Alhoewel, differensiaal oorvloed toetse wys dat daar 4-5 vou verskillende in die oorvloed van Pasteurellaceae en Prevotellaceae tussen die TB gevalle en kontrole groep. Daar was boonop geen merkwaardige verskille in die mikrobiotaprofiele of alfa diversiteite in TB gevalle wat TB behandeling ontvang het by 2 of 6 maande nie. Alhoewel, differensiaal oorvloed toetse het ʼn vermindering in die oorvloed van Veillonellaceae, Staphylococcaceae, Prevotellaceae, Neisseriaceae, Enterobacteriaceae and Aerococcaceae identifiseer in die TB gevalle na TB behandeling. Gevolgtrekking:Hierdie ondersoek het geen merkwaardige verskille tussen die respiratoriese patogene in kinders met en sonder PTB waargeneem nie. Ingelyks, geen verskille in alfa en beta diversiteit tussen die respiratoriese mikrobiota van TB gevalle en kontrole, of na TB behandeling was waargeneem nie. Alhoewel verskille in die oorvloed van sommige gesinne, tussen TB gevalle en kontrolle by basislyn, en voor en na TB behandeling voorstel dat verder navorsing gedoen moet word op hierdie onderwerp aagesien dat daar baie beperkings was wat die bevinding kon beïnvloed. Hierdie studie het bygedra tot die tekort aan beskikbare data met betrekking tot die respiratoriese mikrobiota in kinders met moontlike PTB in ‘n TB endemiese omgewing en het die uitdagings van die uitvoer van mikrobioom navorsing in hulpbron-beperkte omgewings uitgelig.

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