Molecular detection of Mycobacterium tuberculosis in stool of children with suspected intrathoracic tuberculosis

Bosch, Corne (2018-03)

Thesis (MMedSc)--Stellenbosch University, 2018.

Thesis

ENGLISH SUMMARY: The bacteriological confirmation of tuberculosis in children is challenging. The current diagnostic gold standard, liquid culture of respiratory specimens, has low sensitivity in paucibacillary paediatric tuberculosis, and sputum collection in young children is relatively invasive and resource-intensive. Stool is easy to collect and may contain mycobacterial deoxyribonucleic acid (DNA) from swallowed sputum. However, the performance of polymerase chain reaction (PCR) assays, including Xpert MTB/RIF and HAIN FluoroType may be affected by PCR inhibition from stool enzymes and by instrument failure due to particulate matter blocking filters. In this study, we aimed to evaluate the diagnostic performance of stool specimens using a variety of stool pre-processing steps, including decontamination and lyophilisation; as well as various DNA extraction and molecular detection protocols. This study formed part of a larger prospective study involving children with suspected intrathoracic tuberculosis where up to 6 respiratory specimens were collected. Stool specimens were collected at enrolment where one portion was tested by a direct Xpert MTB/RIF protocol; the second portion was frozen for lyophilisation and/or DNA extraction protocols followed by PCR-based molecular detection. DNA was extracted from stools using either a manual commercial stool or soil kit. Extracted DNA was tested for the presence of mycobacterial DNA using the Xpert MTB/RIF cartridge according to standard manufacturer’s protocol and/or a modified “Tube Fill” protocol; and/or the HAIN FluoroType® MTB assay. The results were compared to a composite reference standard and a secondary reference standard (first respiratory culture), which was a better reflection of true performance in our setting. Our results indicate that the standard and Tube Fill Xpert MTB/RIF protocols, as well as the FluoroType MTB detection platforms are able to detect mycobacterial DNA from stool specimens. The Xpert MTB/RIF performed directly on decontaminated stool specimens was found to have the best diagnostic accuracy with sensitivities of 45.8% - 47.1% and specificities of 97.8% - 98.2%. This method was also found to have the lowest indeterminate rate of 3.4% - 10.3%. The other protocols investigated displayed unacceptable sensitivity and specificity combinations with high rates of indeterminate results. The high indeterminate rates were concerning and further optimisation and method simplification are required to propose stool as a non-invasive specimen type for the rapid confirmation of TB in children.

AFRIKAANSE OPSOMMING: Die bakteriologiese bevestiging van tuberkulose in kinders is uitdagend. Kultuur van die organisme, Mycobacterium tuberculosis, is die huidige goue-standaardtoets vir diagnose van tuberkulose. Ongelukkig is die kultuur van respiratoriese monsters in pediatriese pasiënte vermoeilik, aangesien die laer organismelading van pediatriese tuberkulose en die probleme met sputum versameling in jong kinders. Ontlasting (stoelgang) is maklik om te versamel en kan moontlik mikobakteriële deoksiribonukleïensuur (DNS) bevat vanaf die ingeslukte sputum. Die sukses van die polymerase ketting reaksie (PKR)-gebaseerde toetse, insluitend die Xpert MTB/RIF en die HAIN FluoroType kan egter nadelig beinvloed word deur PKR-inhibeerders teenwoordig in stoelgang (bv. ensieme), asook apparaat wanfunksionering as gevolg van stoelgang restes wat die filtreerders blok. Die doel van hierdie studie was om die diagnostiese benut van stoelgang as monstertipe vir die diagnose van tuberkulose te bepaal. Verskeie stoelgangs voorbereiding stappe, insluitend dekontaminasie en vriesdroging; asook verskeie DNS ekstraksie en molekulêre opsporingsmetodes is ondersoek. Die studie was deel van ‘n omvattende studie wat tot en met 6 respiratoriese monsters van kinders met vermoede pulmonale tuberkulose geneem het. Vir die doel van ons studie, is stoelgang monsters aan die begin van die studie versamel, en een porsie is deurmiddel van ‘n Xpert MTB/RIF getoets en ʼn tweede porsie is gevries vir latere vriesdroging gevolg deur DNS ekstraksie en PKR-gebaseerde molekulêre opsporing. DNS is geëkstraheer vanaf stoelgang monsters deur die gebruik van ʼn geoutomatiseerde kommersiële stoelgang of grond ekstraksie kit. Geëkstraheerde DNS is getoets vir die teenwoordigheid van mikobakteriële DNS deur gebruik te maak die Xpert MTB/RIF toets volgens die standaard protokol as ook ‘n aangepasde “Tube Fill” tegniek. Die Hain Fluorotype® MTB metode is ook ondersoek. Uitslae van die verskeie metodes is vergelyk met ‘n saamgestelde verwysingstandaard asook ‘n sekondêre verwysingstandaard (die eerste respiratoriese monster), wat ‘n beter besinning is van wat in praktyk in ons omgewing gebeur. Die studieresultate toon aan dat die standaard en “Tube Fill” Xpert MTB/RIF protokole, asook die HAIN FluoroType MTB deteksie platvorm wel mikobakteriële DNS vanaf stoelgang monsters kan opspoor. Die Xpert MTB/RIF gedoen op gedekontamineerde stoelgang het die beste sensitiwiteit (45.8% - 47.1%) en spesifisiteit (97.8% - 98.2%) opgelewer met ‘n onbepaaldheids persentasie van 3.4% - 10.3%. Die sensitiwiteit en spesifisiteit van elk van die ander protokole was nie belowend nie, en die metodes het ook hoë onpebaaldheids syfers getoon. Die hoeveelheid monsters wat nie ‘n resultaat kon oplewer nie was kommerwekkend en verg verdere ondersoek om die tegnieke te verbeter en te vereenvoudig. Verdere studies is dus nodig voor stoelgang as ‘n nie-indringende monstertipe vir die spoedige bevestiging van tuberkulose in kinders voorgestel kan word.

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