Masters Degrees (Medical Microbiology)

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Browsing Masters Degrees (Medical Microbiology) by Author "Bosch, Corne"

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    Molecular detection of Mycobacterium tuberculosis in stool of children with suspected intrathoracic tuberculosis
    (Stellenbosch : Stellenbosch University, 2018-03) Bosch, Corne; Hoek, Kim Gilberte Pauline; Walters, Elisabetta; Warren, Robin; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.
    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.